Inducing cellular immune responses to hepatitis B virus using peptide and nucleic acid compositions

ABSTRACT

This invention uses our knowledge of the mechanisms by which antigen is recognized by T cells to develop epitope-based vaccines directed towards HBV. More specifically, this application communicates our discovery of pharmaceutical compositions and methods of use in the prevention and treatment of HBV infection.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a Continuation-In-Part (“CIP”) of U.S. Ser. No.08/820,360 filed Mar. 12, 1997, which claims the benefit of U.S.Provisional Application No. 60/013,363 filed Mar. 13, 1996 and nowabandoned. The present application is, also a CIP of U.S. Ser. No.09/189,702 filed Nov. 10, 1998, which is a CIP of U.S. Ser. No.08/205,713 filed Mar. 4, 1994, which is a CIP of Ser. No. 08/159,184filed Nov. 29, 1993 and now abandoned, which is a CIP of Ser. No.08/073,205 filed Jun. 4, 1993 and now abandoned, which is a CIP of Ser.No. 08/027,146 filed Mar. 5, 1993 and now abandoned. The presentapplication is also related to U.S. Ser. No. 08/197,484, U.S. Ser. No.08/464,234, U.S. Ser. No. 08/464,496, U.S. Ser. No. 08/464,031,abandoned U.S. Ser. No. 08/464,433, and U.S. Ser. No. 08/461,603, whichis a continuation of abandoned U.S. Ser. No. 07/935,811, which is a CIPof abandoned U.S. Ser. No. 07/874,491, which is a CIP of abandoned U.S.Ser. No. 07/827,682, which is a CIP of abandoned U.S. Ser. No.07/749,568. The present application is also related to U.S. patentapplication entitled “Peptides and Methods for Creating SyntheticPeptides with Modulated Binding Affinity for HLA Molecules”, AttorneyDocket No. 018623-009520, filed Jan. 6, 1999, which is a CIP of U.S.Ser. No. 08/815,396, which is a CIP of abandoned U.S. Ser. No.60/013,113. Furthermore, the present application is related to U.S. Ser.No. 09/017,735, which is a CIP of abandoned U.S. Ser. No. 08/589,108;U.S. Ser. No. 08/753,622, U.S. Ser. No. 08/822,382, abandoned U.S. Ser.No. 60/013,980, U.S. Ser. No. 08/454,033, U.S. Ser. No. 09/116,424, U.S.Ser. No. 08/205,713, and U.S. Ser. No. 08/349,177, which is a CIP ofabandoned U.S. Ser. No. 08/159,184, which is a CIP of abandoned U.S.Ser. No. 08/073,205, which is a CIP of abandoned U.S. Ser. No.08/027,146. The present application is also related to U.S. Ser. No.09/017,524, U.S. Ser. No. 08/821,739, abandoned U.S. Ser. No.60/013,833, U.S. Ser. No. 08/58,409, U.S. Ser. No. 08/589,107, U.S. Ser.No. 08/451,913, U.S. Ser. No. 08/186,266, U.S. Ser. No. 09/116,061, andU.S. Ser. No. 08/347,610, which is a CIP of U.S. Ser. No. 08/159,339,which is a CIP of abandoned U.S. Ser. No. 08/103,396, which is a CIP ofabandoned U.S. Ser. No. 08/027,746, which is a CIP of abandoned U.S.Ser. No. 07/926,666. The present application is also related to U.S.Ser. No. 09/017,743, U.S. Ser. No. 08/753,615; U.S. Ser. No. 08/590,298,U.S. Ser. No. 09/115,400, and U.S. Ser. No. 08/452,843, which is a CIPof U.S. Ser. No. 08/344,824, which is a CIP of abandoned U.S. Ser. No.08/278,634. The present application is also related to provisional U.S.Ser. No. 60/087,192 and U.S. Ser. No. 09/009,953, which is a CIP ofabandoned U.S. Ser. No. 60/036,713 and abandoned U.S. Ser. No.60/037,432. In addition, the present application is related to U.S. Ser.No. 09/098,584 and to Provisional U.S. patent application entitled“Identification of Broadly Reaactive HLA Restricted T Cell Epitopes”,Attorney Docket No. 018623-013800, filed of even date herewith. All ofthe above applications are incorporated herein by reference.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was funded, in part, by the United States governmentunder grants with the National Institutes of Health. The U.S. governmenthas certain rights in this invention.

INDEX

-   I. Background of the Invention-   II. Summary of the Invention-   III. Brief Description of the Figures-   IV. Detailed Description of the Invention    -   A. Definitions    -   B. Stimulation of CTL and HTL responses against HBV    -   C. Immune Response Stimulating Peptides        -   1. Binding Affinity of the Peptides for HLA Molecules        -   2. Peptide Binding Motifs and Supermotifs            -   a) HLA-A1 supermotif            -   b) HLA-A2 supermotif            -   c) HLA-A3 supermotif            -   d) HLA-A24 supermotif            -   e) HLA-B7 supermotif            -   f) HLA-B27 supermotif            -   g) HLA-B44 supermotif            -   h) HLA-B58 supermotif            -   i) HLA-B62 supermotif            -   j) HLA-A1 motif            -   k) HLA-A3 motif            -   1) HLA-A11 motif            -   m) HLA-A24 motif            -   n) HLA-A2.1 motif            -   o) HLA-DR-1-4-7 supermotif            -   p) HLA-DR3 motifs        -   3. Enhancing Population Coverage of the Vaccine    -   D. Immune Response Stimulating Peptide Analogs    -   E. Computer Screening of Protein Sequences from Disease-Related        Antigens for Supermotif or Motif Containing Peptides    -   F. Assays to Detect T-Cell Responses    -   G. Preparation of Peptides    -   H. Use of Peptide Epitopes for Evaluating Immune Responses    -   I. Vaccine Compositions        -   1. Minigene Vaccines        -   2. Combinations with Helper Peptides    -   J. Administration of Vaccines for Therapeutic or Prophylactic        Purposes    -   K. Kits-   V. Examples

I. BACKGROUND OF THE INVENTION

Chronic infection by hepatitis B virus (HBV) affects at least 5% of theworld's population and is a major cause of cirrhosis and hepatocellularcarcinoma (Hoofnagle, J., N. Engl. J. Med. 323:337, 1990; Fields, B. andKnipe, D., In: Fields Virology 2:2137, 1990). The World HealthOrganization lists hepatitis B as a leading cause of death worldwide,close behind chronic pulmonary disease, and more prevalent than AIDS.Chronic HBV infection can range from an asymptomatic carrier state tocontinuous hepatocellular necrosis and inflammation, and can lead tohepatocellular carcinoma.

The immune response to HBV is believed to play an important role incontrolling hepatitis B infection. A variety of humoral and cellularresponses to different regions of the HBV nucleocapsid core and surfaceantigens have been identified. T cell mediated immunity, particularlyinvolving class I human leukocyte antigen-restricted cytotoxic Tlymphocytes (CTL), is believed to be crucial in combatting establishedHBV infection.

Class I human leukocyte antigen (HLA) molecules are expressed on thesurface of almost all nucleated cells. CTL recognize peptide fragments,derived from intracellular processing of various antigens, in the formof a complex with class I HLA molecules. This recognition event thenresults in the destruction of the cell bearing the HLA-peptide complexdirectly or the activation of non-destructive mechanisms e.g., theproduction of interferon, that inhibit viral replication.

Several studies have emphasized the association between self-limitingacute hepatitis and multispecific CTL responses (Penna, A. et al., J.Exp. Med. 174:1565, 1991; Nayersina, R. et al., J. Immunol. 150:4659,1993). Spontaneous and interferon-related clearance of chronic HBVinfection is also associated with the resurgence of a vigorous CTLresponse (Guidotti, L. G. et al., Proc. Natl. Acad. Sci. USA 91:3764,1994). In all such cases the CTL responses are polyclonal, and specificfor multiple viral proteins including the HBV envelope, core andpolymerase antigens. By contrast, in patients with chronic hepatitis,the CTL activity is usually absent or weak, and antigenicallyrestricted.

The crucial role of CTL in resolution of HBV infection has been furtherunderscored by studies using HBV transgenic mice. Adoptive transfer ofHBV-specific CTL into mice transgenic for the HBV genome resulted insuppression of virus replication. This effect was primarily mediated bya non-lytic, lymphokine-based mechanism (Guidotti, L. G. et al., Proc.Natl. Acad. Sci. USA 91:3764, 1994; Guidotti, L. G., Guilhot, S., andChisari, F. V. J. Virol. 68:1265, 1994; Guidotti, L. G. et al., J.Virol. 69:6158, 1995; Gilles, P. N., Fey, G., and Chisari, F. V., J.Virol. 66:3955, 1992).

As is the case for HLA class I restricted responses, HLA class IIrestricted T cell responses are usually detected in patients with acutehepatitis, and are absent or weak in patients with chronic infection(Chisari, F. V. and Ferrari, C., Annu. Rev. Immunol. 1′:29, 1995). HLAClass II responses are tied to activation of helper T cells (IT Ls)Helper T lymphocytes, which recognize Class II HLA molecules, maydirectly contribute to the clearance of HBV infection through thesecretion of cytokines which suppress viral replication (Franco, A. etal., J. Immunol. 159:2001, 1997). However, their primary role in diseaseresolution is believed to be mediated by inducing activation andexpansion of virus-specific CTL and B cells.

In view of the heterogeneous immune response observed with HBVinfection, induction of a multi-specific cellular immune responsedirected simultaneously against multiple epitopes appears to beimportant for the development of an efficacious vaccine against HBV.There is a need to establish vaccine embodiments that elicit immuneresponses that correspond to responses seen in patients that clear HBVinfection. Epitope-based vaccines appear useful.

Upon development of appropriate technology, the use of epitope-basedvaccines has several advantages over current vaccines. The epitopes forinclusion in such a vaccine are to be selected from conserved regions ofviral or tumor-associated antigens, in order to reduce the likelihood ofescape mutants. The advantage of an epitope-based approach over the useof whole antigens is that there is evidence that the immune response towhole antigens is directed largely toward variable regions of theantigen, allowing for immune escape due to mutations. Furthermore,immunosuppressive epitopes that may be present in whole antigens can beavoided with the use of epitope-based vaccines.

Additionally, with an epitope-based vaccine approach, there is anability to combine selected epitopes (CTL and HTL) and additionally tomodify the composition of the epitopes, achieving, for example, enhancedimmunogenicity. Accordingly, the immune response can be modulated, asappropriate, for the target disease. Similar engineering of the responseis not possible with traditional approaches.

Another major benefit of epitope-based immune-stimulating vaccines istheir safety. The possible pathological side effects caused byinfectious agents or whole protein antigens, which might have their ownintrinsic biological activity, is eliminated.

An epitope-based vaccine also provides the ability to direct and focusan immune response to multiple selected antigens from the same pathogen.Thus, patient-by-patient variability in the immune response to aparticular pathogen may be alleviated by inclusion of epitopes frommultiple antigens from that pathogen in a vaccine composition. A“pathogen” may be an infectious agent or a tumor associated molecule.

However, one of the most formidable obstacles to the development ofbroadly efficacious epitope-based immunotherapeutics has been theextreme polymorphism of HLA molecules. To date, effectivenon-genetically biased coverage of a population has been a task ofconsiderable complexity; such coverage has required that epitopes beused specific for HLA molecules corresponding to each individual HLAallele, therefore, impractically large numbers of epitopes would have tobe used in order to cover ethnically diverse populations. There hasexisted a need to develop peptide epitopes that are bound by multipleHLA antigen molecules for use in epitope-based vaccines. The greater thenumber of HLA antigen molecules bound, the greater the breadth ofpopulation coverage by the vaccine.

Furthermore, as described herein in greater detail, a need has existedto modulate peptide binding properties, for example so that peptidesthat are able to bind to multiple HLA antigens do so with an affinitythat will stimulate an immune response. Identification of epitopesrestricted by more than one HLA allele at an affinity that correlateswith immunogenicity is important to provide thorough populationcoverage, and to allow the elicitation of responses of sufficient vigorwhereby the natural immune responses noted in self-limiting acutehepatitis, or of spontaneous clearance of chronic HBV infection isinduced in a diverse segment of the population. Such a response can alsotarget a broad array of epitopes. The technology disclosed hereinprovides for such favored immune responses.

The information provided in this section is intended to disclose thepresently understood state of the art as of the filing date of thepresent application. Information is included in this section which wasgenerated subsequent to the priority date of this application.Accordingly, background in this section is not intended, in any way, todelineate the priority date for the invention.

II. SUMMARY OF THE INVENTION

This invention applies our knowledge of the mechanisms by which antigenis recognized by T cells, for example, to develop epitope-based vaccinesdirected towards HBV. More specifically, this application communicatesour discovery of specific epitope pharmaceutical compositions andmethods of use in the prevention and treatment of HBV infection.

An embodiment of the present invention includes a peptide composition ofless than 100 amino acid residues comprising a peptide epitope usefulfor inducing an immune response against hepatitis B virus (HBV) saidepitope (a) having an amino acid sequence of about 8 to about 13 aminoacid residues that have at least 65% identity with a native amino acidsequence for HBV, and, (b) binding to at least one MHC class I HLAallele with a dissociation constant of less than about 500 nM. Further,the peptide composition may comprise an amino acid sequence of at least77% identity, or at least 100% identity with a native HBV amino acidsequence. In a preferred embodiment, the peptide is one of the peptidesdesignated as being from the envelope, polymerase, protein X, ornucleocapsid core regions of HBV. Preferred peptides are described inTables VI through XVII or XXI.

An additional embodiment of the present invention comprises acomposition of less than 100 amino acid residues comprising a peptideepitope useful for inducing an immune response against hepatitis B virus(HBV) said peptide (a) having an amino acid sequence of about 8 to about13 amino acid residues and (b) bearing one of the HLA supernotifs ormotifs set out in Tables I and II. Furthermore, the composition maycomprise a peptide wherein the peptide is one of those described inTables VI through XVII or Table XXI which bear an HLA A1, A2, A3, A24,B7, B27, B44, B58, or B62 supermotif; or an HLA A1, A3, A11, A24, orA2.1 motif or an HLA A*3301, A*3101, A*6801, B*0702, B*3501, B51,B*5301, B*5401 motif.

In one embodiment of a peptide comprising an HLA A2.1 motif, the peptidedoes not bear an L or M at position 2 and V at the C-terminal position 9of a 9 amino acid peptide.

An alternative embodiment of the invention comprises an analog of an HBVpeptide of less than 100 amino acid residues in length that bears an HLAbinding motif, the analog bearing the same HLA binding motif as thepeptide but comprising at least one anchor residue that is differentfrom that of the peptide. In a preferred embodiment, said peptide is ananalog of a peptide described in Table VI through Table XVII bearing anHLA A1, A2, A3, A24, B7, B27, B44, B58, or B62 supermotif; or an HLA A1,A3, A11, A24, or A2.1 motif or A3301, A3101, A6801, B0702, B3501, B51,B5301, B5401 motif.

Embodiments of the invention further include a composition of less than100 amino acid residues comprising a peptide epitope useful for inducingan immune response against hepatitis B virus (HBV) said peptide (a)having an amino acid sequence of about 9 to about 25 amino acid residuesthat have at least 65% identity with a native amino acid sequence forHBV and (b) binding to at least one MHC class II HLA allele with adissociation constant of less than about 1000 nM. In a preferredembodiment, the composition comprises a peptide that has at least 77%,or, 100% identity with a native HBV amino acid sequence. Further, thecomposition may comprise a peptide wherein said peptide is one of thosepeptides described in Table XVIII or Table XIX.

The invention also includes a peptide composition of less than 100 aminoacid residues, said composition comprising an epitope useful forinducing an immune response against hepatitis B virus (HBV) said epitope(a) having an amino acid sequence of about 10 to about 20 amino acidresidues and (b) bearing one of the class II HLA motifs set out in TableIII. In a preferred embodiment, said peptide is one of those peptidesdescribed in Table XVIII or XIX.

Additional embodiments of the invention include a composition thatcomprises an isolated nucleic acid sequence that encodes one of thepeptides set out in Tables VI through XIX or XXI or XXIII.

Alternatively, an embodiment of the invention comprises a compositionthat comprises at least two peptides, at least one of said at least twopeptides selected from Tables VI-XIX or XXI or XXIII. In a preferredembodiment, two or more of the at least two peptides are depicted inTables VI-XIX or XXI or XXIII. The composition may further comprise atleast one nucleic acid sequence. In a preferred embodiment each of saidat least two peptides are encoded by a nucleic acid sequence, whereineach of the nucleic acid sequences are located on a single vector.

Embodiments of the invention additionally include a peptide compositionof less than 100 amino acid residues, said composition comprising anepitope useful for inducing an immune response against HBV, said epitopehaving at least one of the amino acid sequences set out in Table XXIII.

An alternative modality for defining the peptides in accordance with theinvention is to recite the physical properties, such as length; primary,secondary and/or tertiary structure; or charge, which are correlatedwith binding to a particular allele-specific HLA molecule or group ofallele-specific HLA molecules. A further modality for defining peptidesis to recite the physical properties of an HLA binding pocket, orproperties shared by several allele-specific HLA binding pockets (e.g.pocket configuration and charge distribution) and reciting that thepeptide fits and binds to said pocket or pockets.

An additional embodiment of the invention comprises a method forinducing a cytotoxic T cell response to HBV in a mammal comprisingadministering to said mammal at least one peptide from Tables VI to XIXor Table XXI.

Further embodiments of the invention include a vaccine for treating HBVinfection that induces a protective immune response, wherein saidvaccine comprises at least one peptide selected from Tables VI to TableXIX or Table XXI in a pharmaceutically acceptable carrier.

Also included as an embodiment of the invention is a vaccine forpreventing HBV infection that induces a protective immune response,wherein said vaccine comprises at least one peptide selected from TablesVI to XIX or Table XXI in a pharmaceutically acceptable carrier.

The invention further includes an embodiment comprising a method forinducing a cytotoxic T cell response to HBV in a mammal, comprisingadministering to said mammal a nucleic acid sequence encoding a peptideselected from s VI to XIX or Table XXI.

A further embodiment of the invention comprises a kit for a vaccine fortreating or preventing HBV infection, wherein the vaccine induces aprotective immune response, said vaccine comprising at least one peptideselected from Tables VI to XIX or Table XXI in a pharmaceuticallyacceptable carrier and instructions for administration to a patient.

Lastly, the invention includes an embodiment comprising a method formonitoring immunogenic activity of a vaccine for HBV in a patient havinga known HLA-type, the method comprising incubating a T lymphocyte samplefrom the patient with a peptide selected from Tables VI to XIX or TableXXI which binds the product of at least one HLA allele present in saidpatient, and detecting for the presence of a T lymphocyte that binds tothe peptide. In a preferred embodiment, the peptide comprises atetrameric complex.

As will be apparent from the discussion below, other methods andembodiments are also contemplated. Further, novel synthetic peptidesproduced by any of the methods described herein are also part of theinvention.

III. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: FIG. 1 Illustrates the Position of Peptide Epitopes inExperimental Model Minigene Constructs

IV. DETAILED DESCRIPTION OF THE INVENTION

The peptides and corresponding nucleic acid compositions of the presentinvention are useful for stimulating an immune response to HBV either bystimulating the production of CTL or HTL responses. The peptides, whichare derived directly or indirectly from native HBV amino acid sequences,are able to bind to HLA molecules and stimulate an immune response toHBV. The complete polyprotein sequence from HBV and its variants can beobtained from Genbank. Peptides can also be readily determined fromsequence information that may subsequently be discovered for heretoforeunknown variants of HBV as will be clear from the disclosure providedbelow.

The peptides of the invention have been identified in a number of ways,as will be discussed below. Further, analog peptides have been derivedand the binding activity for HLA molecules modulated by modifyingspecific amino acid residues to create peptide analogs exhibitingaltered immunogenicity. Further, the present invention providescompositions and combinations of compositions that enable epitope-basedvaccines that are capable of interacting with multiple HLA antigens toprovide broader population coverage than prior vaccines.

The invention can be better understood with reference to the followingdefinitions:

IV.A. Definitions

“Cross-reactive binding” indicates that a peptide is bound by more thanone HLA molecule; a synonym is degenerate binding.

A “cryptic epitope” elicits a response by immunization with an isolatedpeptide, but the response is not cross-reactive in vitro when intactwhole protein which comprises the epitope is used as an antigen.

A “dominant epitope” is an epitope that induces an immune response uponimmunization with a whole native antigen. (See, e.g., Sercarz, et al.,Annu. Rev. Immunol. 11:729766 (1993)) Such a response is cross-reactivein vitro with an isolated peptide epitope.

With regard to a particular amino acid sequence, an “epitope” is a setof amino acid residues which is involved in recognition by a particularimmunoglobulin, or in the context of T cells, those residues necessaryfor recognition by T cell receptor proteins and/or MajorHistocompatibility Complex (MHC) receptors. In an immune system setting,in vivo or in vitro, an epitope is the collective features of amolecule, such as primary, secondary and tertiary peptide structure, andcharge, that together form a site recognized by an immunoglobulin, Tcell receptor or HLA molecule.

As used herein, “high affinity” with respect to HLA class I molecules isdefined as binding with an IC₅₀ (or K_(D)) of less than 50 nM.“Intermediate affinity” is binding with an IC₅₀ (or K_(D)) of betweenabout 50 and about 500 nM. “High affinity” with respect to binding toHLA class II molecules is defined as binding with an K_(D) of less than100 nM. “Intermediate affinity” is binding with a K_(D) of between about100 and about 1000 nM. Assays for determining binding are described indetail in PCT publications WO 94/20127 and WO 94/03205. Alternatively,binding is expressed relative to a reference peptide. As a particularassay becomes more, or less, sensitive, the IC₅₀'s of the peptidestested may change somewhat. However, the binding relative to thereference peptide will not significantly change. For example, in anassay run under conditions such that the IC₅₀ of the reference peptideincreases 10-fold, the IC₅₀ values of the test peptides will also shiftapproximately 10-fold. Therefore, to avoid ambiguities, the assessmentof whether a peptide is a good, intermediate, weak, or negative binderis generally based on its IC₅₀, relative to the IC₅₀ of a standardpeptide.

“Human Leukocyte Antigen” or “HLA” is a human class I or class II MajorHistocompatibility Complex (MHC) protein (see, Stites, et al.,IMMUNOLOGY, 8^(TH) ED., Lange Publishing, Los Altos, Calif. (1994).

An “HLA supertype or family”, as used herein, describes sets of HLAmolecules grouped on the basis of shared peptide-binding specificities.HLA class I molecules that share somewhat similar binding affinity forpeptides bearing certain amino acid motifs We grouped into HLAsupertypes. The terms HLA superfamily, HLA supertype family, and HLAxx-like supertype molecules (where xx denotes a particular HLA type) aresynonyms.

Throughout this disclosure, results are expressed in terms of “IC₅₀'s.”IC₅₀ is the concentration of peptide in a binding assay at which 50%inhibition of binding of a reference peptide is observed. Given theconditions in which the assays are run (i.e., limiting HLA proteins andlabeled peptide concentrations), these values approximate K_(D) values.It should be noted that IC₅₀ values can change, often dramatically, ifthe assay conditions are varied, and depending on the particularreagents used (e.g., HLA preparation, etc.). For example, excessiveconcentrations of HLA molecules will increase the apparent measured IC₅₀of a given ligand.

The terms “identical” or percent “identity,” in the context of two ormore peptide sequences, refer to two or more sequences or subsequencesthat are the same or have a specified percentage of amino acid residuesthat are the same, when compared and aligned for maximum correspondenceover a comparison window, as measured using a sequence comparisonalgorithms or by manual alignment and visual inspection.

An “immunogenic peptide” or “peptide epitope” is a peptide whichcomprises an allele-specific motif or supermotif such that the peptidewill bind an HLA molecule and induce a CTL and/or HTL response. Thus,immunogenic peptides of the invention are capable of binding to anappropriate HLA molecule and thereafter inducing a cytotoxic T cellresponse, or a helper T cell response, to the antigen from which theimmunogenic peptide is derived.

The phrases “isolated” or “biologically pure” refer to material which issubstantially or essentially free from components which normallyaccompany the material as it is found in its native state. Thus,isolated peptides in accordance with the invention preferably do notcontain materials normally associated with the peptides in their in situenvironment.

“Major Histocompatibility Complex” or “MHC” is a cluster of genes thatplays a role in control of the cellular interactions responsible forphysiologic immune responses. In humans, the MHC complex is also knownas the HLA complex. For a detailed description of the MHC and HLAcomplexes, see, Paul, FUNDAMENTAL IMMUNOLOGY, 3^(RD) ED., Raven Press,New York, 1993.

The term “motif” refers to the pattern of residues in a peptide ofdefined length, usually a peptide of from about 8 to about 13 aminoacids for a class I HLA motif and from about 6 to about 25 amino acidsfor a class II HLA motif, which is recognized by a particular HLAmolecule. Peptide motifs are typically different for each proteinencoded by each human HLA allele and differ in the pattern of theprimary and secondary anchor residues.

A “negative binding residue” is an amino acid which if present atcertain positions (typically not primary anchor positions) of peptideepitope results in decreased binding affinity of the peptide for thepeptide's corresponding HLA molecule.

The term “peptide” is used interchangeably with “oligopeptide” in thepresent specification to designate a series of residues, typicallyL-amino acids, connected one to the other, typically by peptide bondsbetween the α-amino and carboxyl groups of adjacent amino acids. Thepreferred CTL-inducing oligopeptides of the invention are 13 residues orless in length and usually consist of between about 8 and about 11residues, preferably 9 or 10 residues. The preferred HTL-inducingoligopeptides are less than about 50 residues in length and usuallyconsist of between about 6 and about 30 residues, more usually betweenabout 12 and 25, and often between about 15 and 20 residues.

“Pharmaceutically acceptable” refers to a non-toxic, inert, andphysiologically compatible composition.

A “primary anchor residue” is an amino acid at a specific position alonga peptide sequence which is understood to provide a contact pointbetween the immunogenic peptide and the HLA molecule. One to three,usually two, primary anchor residues within a peptide of defined lengthgenerally defines a “motif” for an immunogenic peptide. These residuesare understood to fit in close contact with peptide binding grooves ofan HLA molecule, with their side chains buried in specific pockets ofthe binding grooves themselves. In one embodiment, the primary anchorresidues are located at position 2 (from the amino terminal position)and at the carboxyl terminal position of a 9 residue peptide inaccordance with the invention. The primary anchor positions for eachmotif and supermotif are set forth in Table I. For example, analogpeptides can be created by altering the presence or absence ofparticular residues in these primary anchor positions. Such analogs areused to finely modulate the binding affinity of a peptide comprising aparticular motif or supermotif.

“Promiscuous binding” is where a distinct peptide is recognized by thesame T cell clone in the context of various HLA molecules.

A “protective immune response” refers to a CTL and/or an HTL response toan antigen from an infectious agent or a tumor antigen from which animmunogenic peptide is derived, and thereby preventing or at leastpartially arresting disease symptoms or progression. The immune responsemay also include an antibody response which has been facilitated by thestimulation of helper T cells.

The term “residue” refers to an amino acid or amino acid mimeticincorporated into an oligopeptide by an amide bond or amide bondmimetic.

A “secondary anchor residue” is an amino acid at a position other than aprimary anchor position in a peptide which may influence peptidebinding. A secondary anchor residue occurs at a significantly higherfrequency amongst bound peptides than would be expected by randomdistribution of amino acids at one position. The secondary anchorresidues are said to occur at “secondary anchor positions.” A secondaryanchor residue can be identified as a residue which is present at ahigher frequency among high affinity binding peptides, or a residueotherwise associated with high affinity binding. For example, analogpeptides can be created by altering the presence or absence ofparticular residues in these secondary anchor positions. Such analogsare used to finely modulate the binding affinity of a peptide comprisinga particular motif or supermotif.

A “subdominant epitope” is an epitope which evokes little or no responseupon immunization with whole antigens which comprise the epitope, butfor which a response can be obtained by immunization with an isolatedpeptide, and this response (unlike the case of cryptic epitopes) isdetected when whole protein is used to recall the response in vitro orin vivo.

A “supermotif” is a peptide binding specificity shared by HLA moleculesencoded by two or more HLA alleles. Thus, a preferably is recognizedwith high or intermediate affinity (as defined herein) by two or moreHLA antigens.

“Synthetic peptide” refers to a peptide that is not naturally occurring,but is man-made using such methods as chemical synthesis or recombinantDNA technology.

The nomenclature used to describe peptide compounds follows theconventional practice wherein the amino group is presented to the left(the N-terminus) and the carboxyl group to the right (the C-terminus) ofeach amino acid residue. When amino acid residue positions are referredto in a peptide epitope they are numbered in an amino to carboxyldirection with position one being the position closest to the aminoterminal. In the formulae representing selected specific embodiments ofthe present invention, the amino- and carboxyl-terminal groups, althoughnot specifically shown, are in the form they would assume at physiologicpH values, unless otherwise specified. In the amino acid structureformulae, each residue is generally represented by standard three letteror single letter designations. The L-form of an amino acid residue isrepresented by a capital single letter or a capital first letter of athree-letter symbol, and the D-form for those amino acids having D-formsis represented by a lower case single letter or a lower case threeletter symbol. Glycine has no asymmetric carbon atom and is simplyreferred to as “Gly” or G. Symbols for the amino acids are shown below.Single Letter Symbol Three Letter Symbol Amino Acids A Ala Alanine C CysCysteine D Asp Aspartic Acid E Glu Glutamic Acid F Phe Phenylalanine GGly Glycine H His Histidine I Ile Isoleucine K Lys Lysine L Leu LeucineM Met Methionine N Asn Asparagine P Pro Proline Q Gln Glutamine R ArgArginine S Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan YTyr TyrosineIV.B. Stimulation of CTL and HTL Responses Against HBV

The mechanism by which T cells recognize antigens has been delineatedduring the past ten years. Based on our new understanding of the immunesystem we have generated efficacious peptide epitope vaccinecompositions that can induce a therapeutic or prophylactic immuneresponse to HBV infection in a broad population. For an understanding ofthe value and efficacy of the claimed compositions, a brief review ofthe technology is provided.

A complex of an HLA molecule and a peptidic antigen acts as the ligandrecognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071,1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A., andBodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev.Immunol. 11:403, 1993). Through the study of single amino acidsubstituted antigen analogs and the sequencing of endogenously bound,naturally processed peptides, critical residues that correspond tomotifs required for specific binding to HLA antigen molecules have beenidentified and are described here and set forth in Tables I, II, and III(see also, e.g., Sette, A. and Grey, H. M, Curr. Opin. Immunol. 4:79,1992; Sinigaglia, F. and Hammer, J., Curr. Biol. 6:52, 1994; Engelhard,V. H., Curr. Opin. Immunol. 6:13, 1994). Furthermore, x-raycrystallographic analysis of HLA-peptide complexes has revealed pocketswithin the peptide binding cleft of HLA molecules which accommodateallele-specific residues borne by peptide ligands; these residues inturn determine the HLA binding capacity of the peptides in which theyare present (Brown, J. H. et al., Nature 364:33, 1993; Guo, H. C. etal., Proc. Natl. Acad. Sci. USA 90:8053, 1993; Guo, H. C. et al., Nature360:364, 1992; Silver, M. L. et al., Nature 360:367, 1992; Matsumura, M.et al., Science 257:927, 1992; Madden et al., Cell 70:1035, 1992;Fremont, D. H. et al., Science 257:919, 1992; Saper, M. A., Bjorkman, P.J. and Wiley, D. C., J. Mol. Biol. 219:277, 1991).

Accordingly, the definition of class I and class II allele-specific HLAbinding motifs or class I supermotifs allows identification of regionswithin a protein that have the potential of binding particular HLAantigens (see also e.g., Sette, A. and Grey, H. M., Curr. Opin. Immunol.4:79, 1992; Sinigaglia, F. and Hammer, J., Curr. Biol. 6:52, 1994;Engelhard, V. H., Curr. Opin. Immunol. 6:13, 1994Kast, W. M. et al., J.Immunol., 152:3904, 1994).

Furthermore, a variety of assays to detect and quantify the affinity ofinteraction between peptide and HLA have also been established (Sette,A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992; Sinigaglia, F. andHammer, J., Curr. Biol. 6:52, 1994; Engelhard, V. H., Curr. Opin.Immunol. 6:13, 1994).

We have found that the correlation of binding affinity withimmunogenicity is an important factor to be considered when evaluatingcandidate peptides. Thus, by a combination of motif searches andHLA-peptide binding assays, candidates for epitope-based vaccines havebeen identified. After determining their binding affinity, additionalconfirmatory work can be performed to select, amongst these vaccinecandidates, epitopes with desired characteristics in terms ofantigenicity and immunogenicity. Various strategies can be utilized toevaluate immunogenicity, including:

1) Primary T cell cultures from normal individuals (Wentworth, P. A. etal., Mol. Immunol. 32:603, 1995; Celis, E. et al., Proc. Natl. Acad.Sci. USA 91:2105, 1994; Tsai, V. et al., J. Immunol. 158:1796, 1997;Kawashima, I. et al., Human Immunol. 59:1, 1998); This procedureinvolves the stimulation of PBL from normal subjects with a test peptidein the presence of antigen presenting cells in vitro over a period ofseveral weeks. T cells specific for the peptide become activated duringthis time and are detected using a ⁵¹Cr-release assay involving peptidesensitized target cells.

2) Immunization of HLA transgenic mice (Wentworth, P. A. et al., J.Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol. 8:651,1996; Alexander, J. et al., J. Immunol. 159:4753, 1997); In this method,peptides in incomplete Freund's adjuvant are administered subcutaneouslyto HLA transgenic mice. Several weeks following immunization,splenocytes are removed and cultured in vitro in the presence of testpeptide for approximately one week. Peptide-specific T cells aredetected using a ⁵¹Cr-release assay involving peptide sensitized targetcells and target cells expressing endogenously generated antigen.

3) Demonstration of recall T cell responses from immune individuals whohave recovered from infection, and/or from chronically infected patients(Rehermann, B. et al., J. Exp. Med. 181:1047, 1995; Doolan, D. L. etal., Immunity 7:97, 1997; Bertoni, R. et al., J. Clin. Invest. 100:503,1997; Threlkeld, S. C. et al., J. Immunol. 159:1648, 1997; Diepolder, H.M. et al., J. Virol. 71:6011, 1997). In applying this strategy, recallresponses were detected by culturing PBL from subjects that had beennaturally exposed to the antigen, for instance through infection, andthus had generated an immune response “naturally”. PBL from subjectswere cultured in vitro for 1-2 weeks in the presence of test peptideplus antigen presenting cells (APC) to allow activation of “memory” Tcells, as compared to “naive” Tcells. At the end of the culture period,T cell activity is detected using assays for T cell activity including⁵¹Cr release involving peptide-sensitized targets, T cell proliferationor lymphokine release.

The following describes the peptide epitopes and corresponding nucleicacids of the invention.

IV.C. Immune Response Stimulating Peptides

As indicated herein, the large degree of HLA polymorphism is animportant factor to be taken into account with the epitope-basedapproach to vaccine development. To address this factor, epitopeselection encompassing identification of peptides capable of binding athigh or intermediate affinity to multiple HLA molecules is preferablyutilized, most preferably these epitopes bind at high or intermediateaffinity to two or more allele specific HLA molecules.

IV.C.1. Binding Affinity of the Peptides for HLA Molecules

CTL-inducing peptides of interest for vaccine compositions preferablyinclude those that have a binding affinity for class I HLA molecules ofless than 500 nM. HTL-inducing peptides preferably include those thathave a binding affinity for class II HLA molecules of less than 1000 nM.For example, peptide binding is assessed by testing the capacity of acandidate peptide to bind to a purified HLA molecule in vitro. Peptidesexhibiting high or intermediate affinity are then considered for furtheranalysis. Selected peptides are tested on other members of the supertypefamily. In preferred embodiments, peptides that exhibit cross-reactivebinding preferably are then used in cellular screening analyses. Apeptide is considered to be an epitope if it possesses the molecularfeatures that form the binding site for a particular immunoglobulin or Tcell receptor protein.

As disclosed herein, high HLA binding affinity is correlated withgreater immunogenicity. Greater immunogenicity can be manifested inseveral different ways. Immunogenicity corresponds to whether an immuneresponse is elicited at all, and to the vigor of any particularresponse. For example, a peptide might elicit an immune response in adiverse array of the population, yet in no instance produce a vigorousresponse. In accordance with these principles, close to 90% of highbinding peptides have been found to be immunogenic, as contrasted withabout 50% of the peptides which bind with intermediate affinity.Moreover, higher binding affinity peptides leads to more vigorousimmunogenic responses. As a result, less peptide is required to elicit asimilar biological effect if a high affinity binding peptide is used.Thus, in preferred embodiments of the invention, high binding epitopesare particularly desired.

The relationship between binding affinity for HLA class I molecules andimmunogenicity of discrete peptide epitopes on bound antigens has beendetermined for the first time in the art by the present inventors. Thecorrelation between binding affinity and immunogenicity was analyzed intwo different experimental approaches (Sette, et al., J. Immunol.153:5586-5592, 1994). In the first approach, the immunogenicity ofpotential epitopes ranging in HLA binding affinity over a 10,000-foldrange was analyzed in HLA-A*0201 transgenic mice. In the secondapproach, the antigenicity of approximately 100 different hepatitis Bvirus (HBV)-derived potential epitopes, all carrying A*0201 bindingmotifs, was assessed by using PBL (peripheral blood lymphocytes) ofacute hepatitis patients. Pursuant to these approaches, it wasdetermined that an affinity threshold of approximately 500 nM(preferably 500 nM or less) determines the capacity of a peptide epitopeto elicit a CTL response. These data are true for class I bindingaffinity measurements for naturally processed peptides and forsynthesized T cell epitopes. These data also indicate the important roleof determinant selection in the shaping of T cell responses.

An affinity threshold associated with immunogenicity in the context ofHLA class II DR molecules has also been delineated (Southwood et al. J.Immunology 160:3363-3373,1998, and U.S. Ser. No. 60/087,192 filed May29, 1998). In order to define a biologically significant threshold of DRbinding affinity, a database of the binding affinities of 32DR-restricted epitopes for their restricting element was compiled. Inapproximately half of the cases (15 of 32 epitopes), DR restriction wasassociated with high binding affinities, i.e. binding affinities of lessthan 100 nM. In the other half of the cases (16 of 32), DR restrictionwas associated with intermediate affinity (binding affinities in the100-1000 nM range). In only one of 32 cases was DR restrictionassociated with an IC₅₀ of 1000 nM or greater. Thus, 1000 nM can bedefined as an affinity threshold associated with immunogenicity in thecontext of DR molecules.

The binding affinity of peptides for HLA molecules can be determined asdescribed in Example 1, below.

IV.C.2. Peptide Binding Motifs and Supermotifs

In the past few years evidence has accumulated to demonstrate that alarge fraction of HLA class I, and possibly class II molecules can beclassified into a relatively few supertypes characterized by largelyoverlapping peptide binding repertoires, and consensus structures of themain peptide binding pockets. Through the study of single amino acidsubstituted antigen analogs and the sequencing of endogenously bound,naturally processed peptides, critical residues required forallele-specific binding to HLA molecules have been identified. Thesemotifs are relevant since they indicate peptides that have bindingaffinity for HLA molecules.

For HLA molecule pocket analyses, the residues comprising the B and Fpockets of HLA class I molecules as described in crystallographicstudies (Guo, H. C. et al., Nature 360:364, 1992; Saper, M. A.,Bjorkman, P. J. and Wiley, D. C., J. Mol. Biol. 219:277, 1991; Madden,D. R., Garboczi, D. N. and Wiley, D. C., Cell 75:693, 1993), have beencompiled from the database of Parham, et al. (Parham, P., Adams, E. J.,and Arnett, K. L., Immunol. Rev. 143:141, 1995). In these analyses,residues 9, 45, 63, 66, 67, 70, and 99 were considered to make up the Bpocket, and to determine the specificity for the residue in the secondposition of peptide ligands. Similarly, residues 77, 80, 81, and 116were considered to determine the specificity of the F pocket, and todetermine the specificity for the C-terminal residue of a peptide ligandbound by the HLA molecule.

Peptides of the present invention may also include epitopes that bind toMHC class II DR molecules. A significant difference between class I andclass II HLA molecules is that, although a stringent size restrictionexists for peptide binding to class I molecules, a greater degree ofheterogeneity in both sizes and binding frame positions of the motif,relative to the N and C termini of the peptide, can be demonstrated forclass II peptide ligands. This increased heterogeneity is due to thestructure of the class II-binding groove which, unlike its class Icounterpart, is open at both ends. Crystallographic analysis ofDRB*0101-peptide complexes (see, e.g., Madden, D. R. Ann. Rev. Immunol.13:587 (1995)) showed that the residues occupying position 1 andposition 6 of peptides complexed with DRB*0101 engage two complementarypockets on the DRBa*0101 molecules, with the P1 position correspondingto the most crucial anchor residue and the deepest hydrophobic pocket.Other studies have also pointed to the P6 position as a crucial anchorresidue for binding to various other DR molecules.

Thus, peptides of the present invention are identified by any one ofseveral HLA-specific amino acid motifs. If the presence of the motifcorresponds to the ability to bind several allele-specific LLA antigensit is referred to as a supermotif. The allele-specific HLA moleculesthat bind to peptides that possess a particular amino acid supermotifare collectively referred to as an HLA “supertype.”

The peptide motifs and supermotifs described below provide guidance forthe identification and use of peptides in accordance with the invention.Examples of peptide epitopes bearing the respective supermotif or motifare included in Tables as designated in the description of each motif orsupermotif. The Tables include a binding affinity ratio listing for someof the peptide epitopes. The ratio may be converted to IC₅₀ by using thefollowing formula: IC₅₀ of the standard peptide/ratio=IC₅₀ of the testpeptide (i.e. the peptide epitope). The IC₅₀ values of standard peptidesused to determine binding affinities for Class I peptides are shown inTable IV. The IC₅₀ values of standard peptides used to determine bindingaffinities for Class II peptides are shown in Table V. The peptides usedas standards for the binding assay are examples of standards;alternative standard peptides can also be used when performing such ananalysis.

To obtain the peptide epitope sequences listed in each Table, proteinsequence data from twenty HBV strains (HPBADR, HPBADR1CG, HPBADRA,HPBADRC, HPBADRCG, HPBCGADR, HPBVADRM, HPBADW, HPBADW1, HPBADW2,HPBADW3, HPBADWZ, HPBHEPB, HPBVADW2, HPBAYR, HPBV, HPBVAYWC, HPBVAYWCI,NAD HPBVAYWE) were evaluated for the presence of the designatedsupermotif or motif. Peptide epitopes were also selected on the basis oftheir conservancy. A criterion for conservancy requires that the entiresequence of a peptide be totally conserved in 75% of the sequencesavailable for a specific protein. The percent conservancy of theselected peptide epitopes is indicated on the Tables. The frequency,i.e. the number of strains of the 20 strains in which the peptidesequence was identified, is also shown. The “1^(st) position” column inthe Tables designates the amino acid position of the HBV polyproteinthat corresponds to the first amino acid residue of the epitope.Preferred peptides are designated by an asterisk.

HLA Class I Motifs Indicative of CTL Inducing Peptide Epitopes:

IV.C.2.a) HLA-A1 Supermotif

The HLA-A1 supermotif is characterized by peptides having a generalmotif of small (T or S) and hydrophobic (L, I, V, M, or F) primaryanchor residues in position 2, and aromatic (Y, F, or W) primary anchorresidues at the C-terminal position The corresponding family of HLAmolecules that bind to the A1 supermotif (the HLA-A1 supertype) includesA*0101, A*2601, A*2602, A*2501, and A*3201. (DiBrino, M. et al., J.Immunol. 151:5930, 1993; DiBrino, M. et al., J. Immunol. 152:620, 1994;Kondo, A. et al., Immunogenetics 45:249, 1997; Dumrese et al.,submitted). Peptides binding to each of the individual HLA proteins canbe modulated by substitutions at primary anchor positions.

Representative peptide epitopes that contain the A1 supermotif are setforth on the attached Table VI.

IV.C.2.b) HLA-A2 Supermotif

The HLA-A2 supermotif is characterized by the presence in peptideligands of small or aliphatic amino acids (L, I, V, M, A, T, or Q) atposition 2 and L, I, V, M, A, or T at the C-terminal position. Thesepositions ate referred to as primary anchors. The corresponding familyof HLA molecules (the HLA-A2 supertype that binds these peptides) iscomprised of at least nine HLA-A proteins: A*0201, A*0202, A*0203,A*0204, A*0205, A*0206, A*0207, A*6802, and A*6901. As explained indetail below, binding to each of the individual allele-specific HLAmolecules can be modulated by substitutions at the primary anchor and/orsecondary anchor positions.

Representative peptide epitopes that contain the A2 supermotif are setforth on the attached Table VII.

IV.C.2.c) HLA-A3 Supermotif

The HLA-A3 supermotif is characterized by peptide ligands having primaryanchor residues: A, L, I, V, M, S, or, T at position 2, and positivelycharged residues, such as R or K at the C-terminal position (in position9 of 9-mers). Exemplary members of the corresponding HLA family of HLAmolecules (the HLA-A3 superfamily) that bind the A3 supermotif include:A3 (A*0301), A11 (A*1101), A31 (A*3101), A*3301, and A*6801. Otherallele-encoded HLA molecules predicted to be members of the A3superfamily include A34, A66, and A*7401. As explained in detail below,peptide binding to each of the individual allele-specific HLA proteinscan be modulated by substitutions of amino acids at the primary and/orsecondary anchor positions of the peptide.

Representative peptide epitopes that contain the A3 supermotif are setforth on the attached Table VIII.

IV.C.2.d) HLA-A24 Supermotif

The HLA-A24 supermotif is characterized by the presence in peptideligands of an aromatic (F, W, or Y) residue as a primary anchor inposition 2 and a hydrophobic (Y, F, L, I, V, or M) residue as primaryanchor at the C-terminal position. The corresponding family of HLAmolecules that bind to the A24 supermotif (the A24 supertype) includesA*2402, A*3001, and A*2301. Peptide binding to each of theallele-specific HLA molecules can be modulated by substitutions atprimary anchor positions.

Representative peptide epitopes that contain the A24 supermotif are setforth on the attached Table IX.

IV.C.2.e) HLA-B7 Supermotif

The HLA-B7 supermotif is characterized by peptides bearing proline inposition 2 as a primary anchor and hydrophobic or aliphatic amino acids(L, I, V, M, A, F, W, or Y) as the primary anchor at the C-terminalposition. The corresponding family of HLA molecules that bind the B7supermotif (the HLA-B7 supertype) is comprised of at least a dozen HLA-Bproteins including B7, B*3501-1, B*3502-2, B*3501-3, B51, B*5301,B*5401, B*5501, B*5401, B*5501, B*5502, B*5601, B*6701, and B*7801 (See,e.g., Sidney, et al., J. Immunol. 154:247 (1995); Barber, et al., Curr.Biol. 5:179 (1995); Hill, et al., Nature 360:434 (1992); Rammensee, etal., Immunogenetics 41:178 (1995)). As explained in detail below,peptide binding to each of the individual allele-specific HLA proteinscan be modulated by substitutions at the primary and/or secondary anchorpositions of the peptide.

Representative peptide epitopes that contain the B7 supermotif are setforth on the attached Table X.

IV.C.2.f) HLA-B27 Supermotif

The HLA-B27 supermnotif is characterized by the presence in peptideligands of positively charged (R, H, or K) residues as primary anchorsat position 2 and hydrophobic (A, L, I, V, M, Y, F, or W) residues asprimary anchors at the C-terminal. Exemplary members of thecorresponding HLA molecules that bind to the B27 supermotif (the B27supertype) include B*14, B*1509, B*38, B*3901, B*3902, B*73, and variousB27 subtypes. Peptide binding to each of the allele-specific HLAmolecules can be modulated by substitutions at primary anchor positions.

Representative peptide epitopes that contain the B27 supermotif are setforth on the attached Table XI.

IV.C.2.g) HLA-B44 Supermotif

The HLA-B44 supermotif is characterized by the presence in peptideligands of negatively charged (D or E) residues as a primary anchor inposition 2, and hydrophobic residues (F, W, Y, L, I, M V, or A) as aprimary anchor at the C-terminal. Exemplary members of the correspondingfamily of HLA molecules that bind to the B44 supermnotif (the B44supertype) include B*3701, B*4402, B*4403, B60, and B61. Peptide bindingto each of the allele-specific HLA molecules can be modulated bysubstitutions at primary anchor positions.

Representative peptide epitopes that contain the B44 supermotif are setforth on the attached Table XII.

IV.C.2.h) HLA-B58 Supermotif

The HLA-B58 supermotif is characterized by the presence in peptideligands of small aliphatic residues (A, S, or T) as primary anchorresidues at position 2 and aromatic or hydrophobic residues (F, W, Y, L,I, or V) as primary anchor residues at the C-terminal. Exemplary membersof the corresponding HLA molecules that bind to the B58 supermotif (theB58 supertype) include B*1516, B*1517, B*5701, B*5702, and B*58. Peptidebinding to each of the allele-specific HLA molecules can be modulated bysubstitutions at primary anchor positions.

Representative peptide epitopes that contain the B58 supermotif are setforth on the attached Table XIII.

IV.C.2.i) HLA-B62 Supermotif

The HLA-B62 supermotif is characterized by the presence in peptideligands of the polar aliphatic residue Q or the hydrophobic aliphaticresidues (L, V, M, or I) as a primary anchor in position 2 andhydrophobic residues (F, W, Y, M, I, or V) as a primary anchor at theC-terminal position. Exemplary members of the corresponding HLAmolecules that a bind to the B62 supermotif (the B62 supertype) includeB46, B52, B62, B75, and B77. Peptide binding to each of theallele-specific HLA molecules can be modulated by substitutions atprimary anchor positions.)

Representative peptide epitopes that contain the B62 supermotif are setforth on the attached Table XIV.

IV.C.2.j) HLA-A1 Motif

The allele-specific HLA-A1 motif is characterized by the presence inpeptide ligands of T, S, or M as a primary anchor residue at position 2and the presence of Y as a primary anchor residue at the C-terminalposition. Alternatively, a primary anchor residue may be present atposition 3 rather than position 2. This motif is characterized by thepresence of D, E, A, or S as a primary anchor residue in position 3 anda Y as a primary anchor residue at the C-terminus. Peptide binding toHLA A1 can be modulated by substitutions at primary and/or secondaryanchor positions.

Representative peptide epitopes that contain the A1 motif are set forthon the attached Table XV.

IV.C.2.k) HLA-A3 Motif

The allele-specific HLA-A3 motif is characterized by the presence inpeptide ligands of L, M, V, I, S, A, T, F, C, G, or D as a primaryanchor residue at position 2 and the presence of K, Y, R, H, F, or A asthe primary anchor residue at the C-terminal position. Peptide bindingto HLA-A3 can be modulated by substitutions at primary and/or secondaryanchor positions.

Representative peptide epitopes that contain the A3 motif are set forthon the attached Table XVI.

IV.C.2.1) HLA-A11 Motif

The allele-specific HLA-A11 motif is characterized by the presence inpeptide ligands of V, T, M, L, I, S, A, G, N, C, D, or F as a primaryanchor residue in position 2 and K, R, Y, or H as a primary anchorresidue at the C-terminal position. Peptide binding to HLA-A 11 can bemodulated by substitutions at primary and/or secondary anchor positions.

Representative peptide epitopes that contain the A11 motif are set forthon the attached Table XVI; peptides bearing the A3 allele-specific motifare also present in Table XVII. The A11 and A3 motifs have a number ofanchor residues in common, separate tables would provide a number ofredundant entries.

IV.C.2.m) HLA-A24 Motif

The allele-specific HLA-A24 motif is characterized by the presence inpeptide ligands of Y, F, W, or M as a primary anchor residue in position2 and F, L, I, or W as a primary anchor residue at the C-terminalposition. Peptide binding to HLA-A24 molecules can be modulated bysubstitutions at primary and/or secondary anchor positions.

Representative peptide epitopes that contain the A24 motif are set forthon the attached Table XVII.

IV.C.2.n) HLA-A2.1 Motif

The allele-specific HLA-A2.1 motif was first determined to becharacterized by the presence in peptide ligands of L, M, V, I, A or Tas a primary anchor residue in position 2 and, L, V, I, A, or T as aprimary anchor residue at the C-terminal position. The preferred andtolerated residues that characterize the primary anchor positions of theHLA-A2.1 motif are identical to the preferred residue of the A2supermotif. Secondary anchor residues that characterize the A2.1 motifhave additionally been defined as disclosed herein. These are disclosedin Table II. Peptide binding to HLA-A2.1 molecules can be modulated bysubstitutions at primary and/or secondary anchor positions.

Representative peptide epitopes that contain the A2.1 motif are setforth on the attached Table VII. These peptides, which bear the HLA-A2supermotif, also contain secondary anchor residues that arecharacteristic of the HLA-A2.1 motif. In one embodiment, the peptideepitope does not bear an L or M at position 2 and V at the C-terminalposition 9 of a 9-amino acid peptide.

The primary anchor residues of the HLA class I peptide epitopesupermotifs and motifs delineated above are summarized in Table I.Primary and secondary anchor positions are summarized in Table II.

Motifs Indicative of Class II HTL Inducing Peptide Epitopes

IV.C.2.o) HLA DR-1-4-7 Supermotif

Motifs have also been identified for peptides that bind to three commonHLA class II types, HLA DRB1*0401, DRB1*0101, and DRB1*0701. Peptidesbinding to these DR molecules carry a motif characterized by a largearomatic or hydrophobic residue in position 1 (Y, F, W, L, I, V, or M)and a small, non-charged residue in position 6 (S, T, C, AP, V, I, L, orM). Allele specific secondary effects and secondary anchors for each ofthese HLA types have also been identified. These are set forth in TableIII. Peptide binding to HLA-DR4, DR1, and DR7 can be modulated bysubstitutions at primary and/or secondary anchor positions.

Representative peptides are set forth in Table XVIII.

IV.C.2.p) HLA DR3 Motifs

Two alternative motifs characterize peptides that bind to HLA-DR3molecules. In the first motif, a large, hydrophobic residue (I, L, V, M,Y, or F) is present in anchor position 1 and D is present as an anchorat position 4, which is defined as being 3 positions from anchorposition 1 towards the carboxyl terminus regardless of the location ofanchor position 1 in the peptide. Lack of either the large, hydrophobicresidue at anchor position 1, or of the negatively charged or amide-likeanchor residue at position 4 may be compensated for by the presence of apositive charge at position 6 (which is defined as being 5 positionsfrom anchor position 1 towards the carboxyl terminus). Thus for thesecond, alternative motif I, L, V, M, Y, F, or A is present at anchorposition 1; D, N, Q, E, S, or T is present at anchor position 4; and K,R, or H is present at anchor position 6. Peptide binding to HLA-DR3 canbe modulated by substitutions at primary and/or secondary anchorpositions.

Representative peptides are set forth in Table IXX.

IV.C.3. Enhancing Population Coverage of the Vaccine

Vaccines that have broad population coverage are preferred because theyare more commercially viable and generally applicable to the mostpeople. Broad population coverage can be obtained using the peptides ofthe invention (and nucleic acid compositions that encode such peptides)through selecting peptide epitopes that bind to HLA alleles which, whenconsidered in total, are present in most of the population. Table XXlists the overall frequencies of the A2-, A3-, and B7-supertypes invarious ethnicities. Coverage in excess of 80% is achieved with thesemotifs. These results suggest that effective and non-ethnically biasedpopulation coverage is achieved upon use of a limited number ofcross-reactive peptides. Although the population coverage reached withthese three main peptide specificities is high, coverage can be expandedto reach 95% population coverage and above, and more easily achievetruly multispecific responses upon use of additional supermotif orallele-specific motif bearing peptides.

Table XX summarizes the HLA supertypes that have been identified, andindicates an estimate of their combined prevalence in major ethnicgroups. The B44-, A1-, and A24-supertypes are present, on average, inover 25% of the world's major ethnic populations. While less prevalentoverall, the B27-, B58-, and B62 supertypes are each present with afrequency >25% in at least one major ethnic group. The Table indicatesthe population coverage achieved by the A2-, A3-, and B7-supertypes, andthe incremental coverage obtained by the addition of A1-, A24-, andB44-supertypes, or all of the supertypes described herein. As shown, byincluding epitopes from the six most frequent supertypes, an averagepopulation coverage of 99% is obtained for five major ethnic groups.

The data presented herein, together with the previous definition of theA2-, A3-, and B7-supertypes, indicates that all antigens, with thepossible exception of A29, B8, and B46, can be classified into a totalof nine HLA supertypes. Focusing on the six most common supertypesaffords population coverage greater than 98% for all major ethnicpopulations.

IV.D. Immune Response Stimulating Peptide Analogs

Although peptides with suitable cross-reactivity among all alleles of asuperfamily are identified by the screening procedures described above,cross-reactivity is not always complete and in such cases procedures tofurther increase cross-reactivity of peptides can be useful; suchprocedures can also be used to modify other properties of the peptides.Having established the general rules that govern cross-reactivity ofpeptides for HLA alleles within a given motif or supermotif,modification (i.e., analoging) of the structure of peptides ofparticular interest in order to achieve broader (or otherwise modified)HLA binding capacity can be performed. More specifically, peptides whichexhibit the broadest cross-reactivity patterns, (both amongst the knownT cell epitopes, as well as the more extended set of peptides thatcontain the appropriate supermotifs), can be produced in accordance withthe teachings herein.

The strategy employed utilizes the motifs or supermotifs which correlatewith binding to certain HLA molecules. The motifs or supermotifs aredefined by having primary anchors, though secondary anchors can also bemodified. Analog peptides can be created by substituting amino acidsresidues at primary anchor, secondary anchor, or at primary andsecondary anchor positions. Generally, analogs are made for peptidesthat already bear a motif or supermotif. Preferred secondary anchorresidues of supermotifs and motifs that have been defined for HLA classI and class II binding peptides are shown in Tables II and III,respectively.

For a number of the motifs or supermotifs in accordance with theinvention, residues are defined which are deleterious to binding toallele-specific HLA molecules or members of HLA supertypes that bind tothe respective motif or supermotif (Tables II and III). Accordingly,removal of residues that are detrimental to binding can be performed inaccordance with the present invention. For example, in the case of theA3 supertype, when all peptides that have such deleterious residues areremoved from the population of analyzed peptides, the incidence ofcross-reactivity increases from 22% to 37% (see, e.g., Sidney, J. etal., Hu. Immunol. 45:79, 1996). Thus, one strategy to improve thecross-reactivity of peptides within a given supermotif is simply todelete one or more of the deleterious residues present within a peptideand substitute a small “neutral” residue such as Ala (that may notinfluence T cell recognition of the peptide). An enhanced likelihood ofcross-reactivity is expected if, together with elimination ofdetrimental residues within a peptide, residues associated with highaffinity binding to multiple alleles within a superfamily are inserted.

To ensure that changes in the native or original epitope recognized by Tcells do not lead to a failure of killing antigen presenting cellspresenting the unaltered “wild type” peptide (or, in the case of classII epitopes, a failure to elicit helper T cells that cross-react withthe wild type peptides), the variant peptide may be used to immunize Tcells in vitro from individuals of the appropriate HLA allele, and thecells' capacity to induce lysis of wild type peptide sensitized targetcells is evaluated. In both class I and class II systems it will bedesirable to use as targets, cells that have been either infected ortransfected with the appropriate genes to establish whether endogenouslyproduced antigen is also recognized by the relevant T cells.

Another embodiment of the invention to ensure adequate numbers ofcross-reactive cellular binders is to create analogs of weak bindingpeptides. Class I peptides exhibiting binding affinities of 500-50000nM, and carrying an acceptable but suboptimal primary anchor residue atone or both positions can be “fixed” by substituting preferred anchorresidues in accordance with the respective supertype. The analogpeptides can then be tested for crossbinding activity.

Another embodiment for generating effective peptide analogs involves thesubstitution of residues that have an adverse impact on peptidestability or solubility in a liquid environment. This substitution mayoccur at any position of the peptide epitope. For example, a cysteine(C) can be substituted out in favor of α-amino butyric acid. Due to itschemical nature, cysteine has the propensity to form disulfide bridgesand sufficiently alter the peptide structurally so as to reduce bindingcapacity. Substituting α-amino butyric acid for C not only alleviatesthis problem, but actually improves binding and crossbinding capabilityin certain instances (Review: A. Sette et al, In: Persistent ViralInfections, Eds. R. Ahmed and I. Chen, John Wiley & Sons, England, inpress, 1998). Substitution of cysteine with α-amino butyric acid mayoccur at any residue of a peptide epitope, i.e. at either anchor ornon-anchor positions.

In general, CTL and HTL responses are not directed against all possibleepitopes. Rather, they are restricted to a few immunodominantdeterminants (Zinkemagel, et al., Adv. Immunol. 27:5159, 1979; Bennink,et al., J. Exp. Med. 168:19351939, 1988; Rawle, et al., J. Immunol.146:3977-3984, 1991). It has been recognized that immunodominance(Benacerraf, et al., Science 175:273-279, 1972) could be explained byeither the ability of a given epitope to selectively bind a particularHLA protein (determinant selection theory) (Vitiello, et al., J.Immunol. 131:1635, 1983); Rosenthal, et al., Nature 267:156-158, 1977),or being selectively recognized by the existing TCR (T cell receptor)specificity (repertoire theory) (Klein, J., IMMUNOLOGY, THE SCIENCE OFSELFNONSELF DISCRIMINATION, John Wiley & Sons, New York, pp. 270-310,1982). It has been demonstrated that additional factors, mostly linkedto processing events, can also play a key role in dictating, beyondstrict immunogenicity, which of the many potential determinants will bepresented as immunodominant (Sercarz, et al., Annu. Rev. Immunol.11:729-766, 1993).

The concept of dominance and subdominance is relevant to immunotherapyof both infectious diseases and cancer. For example, in the course ofchronic viral disease, recruitment of subdominant epitopes can beimportant for successful clearance of the infection, especially ifdominant CTL or HTL specificities have been inactivated by functionaltolerance, suppression, mutation of viruses and other mechanisms(Franco, et al., Curr. Opin. Immunol. 7:524-531, (1995)). In the case ofcancer and tumor antigens, CTLs recognizing at least some of the highestbinding affinity peptides might be functionally inactivated. Lowerbinding affinity peptides are preferentially recognized at these times.

In particular, it has been noted that a significant number of epitopesderived from known non-viral tumor associated antigens (TAA) bind HLAclass I with intermediate affinity (IC₅₀ in the 50-500 nM range). Forexample, it has been found that 8 of 15 known TAA peptides recognized bytumor infiltrating lymphocytes (TIL) or CTL bound in the 50-500 nMrange. (These data are in contrast with estimates that 90% of knownviral antigens that were recognized as peptides bound HLA with IC₅₀ of50 nM or less, while only approximately 10% bound in the 50-500 nM range(Sette, et al., J. Immunol., 153:558-5592 (1994)). In the cancer settingthis phenomenon is probably due to elimination, or functional inhibitionof the CTL recognizing several of the highest binding peptides,presumably because of T cell tolerization events.

Without intending to be bound by theory, it is believed that because Tcells to dominant epitopes may have been clonally deleted, selectingsubdominant epitopes may allow extant T cells to be recruited, whichwill then lead to a therapeutic response. However, the binding of HLAmolecules to subdominant epitopes is often less vigorous than todominant ones. Accordingly, there is a need to be able to modulate thebinding affinity of particular immunogenic epitopes for one or more BLAmolecules, and thereby to modulate the immune response elicited by thepeptide. Thus a need exists to prepare analog peptides which elicit amore vigorous response. This ability would greatly enhance theusefulness of peptide-based vaccines and therapeutic agents.

Representative analog peptides are set forth in Table XXI. The Tableindicates the length and sequence of the analog peptide as well as themotif or supermotif, if appropriate. The information in the “FixedNomenclature” column indicates the residues substituted at the indicatedposition numbers for the respective analog.

IV.E. Computer Screening of Protein Sequences from Disease-RelatedAntigens for Supermotif or Motif Containing Peptides

Computer programs that allow the rapid screening of protein sequencesfor the occurrence of the subject supermotifs or motifs are encompassedby the present invention; as are programs that permit the generation ofanalog peptides. These programs are implemented to analyze anyidentified amino acid sequence or operate on an unknown sequence andsimultaneously determine the sequence and identify motif-bearingepitopes thereof; analogs can be simultaneously determined as well.Generally, the identified sequences will be from a pathogenic organismor a tumor-associated peptide. For example, the target moleculesconsidered herein include all of the HBV proteins (e.g. surface, core,polymerase, and X).

In cases where the sequence of multiple variants of the same targetprotein are available, peptides are also selected on the basis of theirconservancy. A presently preferred criterion for conservancy definesthat the entire sequence of a peptide be totally conserved in 75% of thesequences evaluated for a specific protein; this definition ofconservancy has been employed herein.

It is important that the selection criteria utilized for prediction ofpeptide binding are as accurate as possible, to correlate mostefficiently with actual binding. Prediction of peptides that bind, forexample, to HLA-A*0201, on the basis of the presence of the appropriateprimary anchors, is positive at about a 30% rate (Ruppert, J. et al.Cell 74:929, 1993). However, by analyzing an extensive peptide-HLAbinding database, the present inventors have developed a number ofallele specific polynomial algorithms that dramatically increase thepredictive value over identification on the basis of the presence ofprimary anchor residues alone. These algorithms take into account notonly the presence or absence of the correct primary anchors, but alsoconsider the positive or deleterious presence of secondary anchorresidues (to account for the impact of different amino acids atdifferent positions). The algorithms are essentially based on thepremise that the overall affinity (or AG) of peptide-HLA interactionscan be approximated as a linear polynomial function of the type:ΔG=a _(1i) ×a _(2i) ×a _(3i) . . . ×a _(ni)

-   -   where ay is a coefficient that represents the effect of the        presence of a given amino acid (i) at a given position (i) along        the sequence of a peptide of n amino acids. An important        assumption of this method is that the effects at each position        are essentially independent of each other. This assumption is        justified by studies that demonstrated that peptides are bound        to HLA molecules and recognized by T cells in essentially an        extended conformation. Derivation of specific algorithm        coefficients has been described in Gulukota et al. (Gulukota, K.        et al., J. Mol. Biol. 267:1258, 1997).

Additional methods to identify preferred peptide sequences, which alsomake use of specific motifs, include the use of neural networks andmolecular modeling programs (Gulukota, K. et al., J. Mol. Biol.267:1258, 1997; Milik et al., Nature Biotechnology 16:753, 1998; Altuviaet al., Hum. Immunol. 58:1, 1997; Altuvia et al, J. Mol. Biol. 249:244,1995).

For example, it has been shown that in sets of A*0201 motif peptides,69% of the peptides containing at least one preferred secondary anchorresidue while avoiding the presence of any deleterious secondary anchorresidues, will bind A*0201 with an IC₅₀ less than 500 nM (Ruppert, J. etal. Cell 74:929, 1993). These algorithms are also flexible in thatcut-off scores may be adjusted to select sets of peptides with greateror lower predicted binding properties, as desired.

In utilizing computer screening to identify peptide epitopes, allprotein sequence or translated sequence may be analyzed using softwaredeveloped to search for motifs, for example the “FINDPATTERNS” program(Devereux, et al. Nucl. Acids Res. 12:387-395, 1984) or MotifSearch 1.4software program (D. Brown, San Diego, Calif.) to identify potentialpeptide sequences containing appropriate HLA binding motifs. Asappreciated by one of ordinary skill in the art a large array ofsoftware and hardware options are available which can be employed toimplement the motifs of the invention relative to known or unknownpeptide sequences. The identified peptides will then be scored usingcustomized polynomial algorithms to predict their capacity to bindspecific HLA class I or class II alleles.

In accordance with the procedures described above, HBV peptides andanalogs thereof that are able to bind HLA supertype groups orallele-specific BLA molecules have been identified (Tables VI-XIX; TableXI).

IV.F. Assays to Detect T-Cell Responses

Once HLA binding peptides are identified, they can be tested for theability to elicit a T-cell response. The preparation and evaluation ofmotif-bearing peptides are described in PCT publications WO 94/20127 andWO 94/03205. Briefly, peptides comprising epitopes from a particularantigen are synthesized and tested for their ability to bind to theappropriate HLA proteins in assays using, for example, purified HLAclass I molecules and radioiodonated peptides and/or cells expressingempty class I molecules (which lack peptide in their receptor) by, forinstance, immunofluorescent staining and flow microfluorimetry,peptide-dependent class I assembly assays, and inhibition of CTLrecognition by peptide competition. Those peptides that bind to theclass I molecule are further evaluated for their ability to serve astargets for CTLs derived from infected or immunized individuals, as wellas for their capacity to induce primary in vitro or in vivo CTLresponses that can give rise to CTL populations capable of reacting withselected target cells associated with a disease. Corresponding assaysare used for evaluation of HLA class II binding peptides.

Conventional assays utilized to detect CTL responses includeproliferation assays, lymphokine secretion assays, direct cytotoxicityassays, and limiting dilution assays. For example, antigen-presentingcells that have been incubated with a peptide can be assayed for theability to induce CTL responses in responder cell populations.Antigen-presenting cells can be normal cells such as peripheral bloodmononuclear cells or dendritic cells. Alternatively, mutant mammaliancell lines that are deficient in their ability to load class I moleculeswith internally processed peptides and that have been transfected withthe appropriate human class I gene may be used to test for the capacityof the peptide to induce in vitro primary CTL responses.

Peripheral blood lymphocytes may be used as the responder cell source ofCTL precursors. The appropriate antigen-presenting cells are incubatedwith peptide and the peptide-loaded antigen-presenting cells are thenincubated with the responder cell population under optimized cultureconditions. Positive CTL activation can be determined by assaying theculture for the presence of CTLs that kill radio-labeled target cells,both specific peptide-pulsed targets as well as target cells expressingendogenously processed forms of the HBV antigen from which the peptidesequence was derived.

More recently, a method has also been devised which allows directquantification of antigen-specific T cells by staining withFluorescein-labelled HLA tetrameric complexes (Altman, J. D. et al.,Proc. Natl. Acad. Sci. USA 90:10330, 1993; Altman, J. D. et al., Science274:94, 1996). Other relatively recent technical developments includestaining for intracellular lymphokines, and interferon release assays orELISPOT assays. Tetramer staining, intracellular lymphokine staining andELISPOT assays all appear to be at least 10-fold more sensitive thanmore conventional assays (Lalvani, A. et al., J. Exp. Med. 186:859,1997; Dunbar, P. R. et al., Curr. Biol. 8:413, 1998; Murali-Krishna, K.et al., Immunity 8:177, 1998).

HTL activation may also be assessed using such techniques as T cellproliferation and secretion of lymphokines, e.g. IL-2.

Alternatively, immunization of HLA transgenic mice can be used todetermine immunogenicity of peptide epitopes. Several transgenic mousemodels including mice with human A2.1, A11, and B7 alleles have beencharacterized and others (e.g., transgenic mice for HLA-A1 and A24) arebeing developed. HLA-DR1 and HLA-DR3 mouse models have also beendeveloped. Additional transgenic mouse models with other HLA alleles maybe generated as necessary. Mice may be immunized with peptidesemulsified in Incomplete Freund's Adjuvant and the resulting T cellstested for their capacity to recognize peptide-pulsed target cells andtarget cells transfected with appropriate genes. CTL responses may beanalyzed using cytotoxicity assays described above. Similarly, HTLresponses may be analyzed using such assays as T cell proliferation orsecretion of lymphokines.

IV.G. Preparation of Peptides

Peptides in accordance with the invention can be prepared synthetically,by recombinant DNA technology, or from natural sources such as nativetumors or pathogenic organisms. Peptide epitopes may be synthesizedindividually or as polyepitopic peptides. Although the peptide willpreferably be substantially free of other naturally occurring host cellproteins and fragments thereof, in some embodiments the peptides may besynthetically conjugated to native fragments or particles.

The peptides in accordance with the invention can be a variety oflengths, and either in their neutral (uncharged) forms or in forms whichare salts. Peptides may be synthesized The peptides in accordance withthe invention are either free of modifications such as glycosylation,side chain oxidation, or phosphorylation; or they contain thesemodifications, subject to the condition that modifications do notdestroy the biological activity of the peptides as described herein.

Desirably, the peptide will be as small as possible while stillmaintaining substantially all of the biological activity of the largepeptide. When possible, it may be desirable to optimize HLA class Ibinding peptides of the invention to a length of about 8 to about 13amino acid residues, preferably 9 to 10. HLA class II binding peptidesmay be optimized to a length of about 6 to about 25 amino acids inlength, preferably to between about 13 and about 20 residues.Preferably, the peptides are commensurate in size with endogenouslyprocessed pathogen-derived peptides or tumor cell peptides that arebound to the relevant HLA molecules. Moreover, the identification andpreparation of peptides of other lengths can be carried out using thetechniques described herein (e.g., the disclosures regarding primary andsecondary anchor positions). However, it is also preferred to identify alarger region of a native peptide that encompasses one and preferablytwo or more epitopes in accordance with the invention. This sequence isselected on the basis that it contains the greatest number of epitopesper amino acid length. It is to be appreciated that epitopes can bepresent in a frame-shifted manner, e.g. a 10 amino acid long peptidecould contain two 9 amino acid long epitopes and one 10 amino acid longepitope; each epitope can be exposed and bound by an HLA molecule uponadministration of a plurality of such peptides. This larger, preferablymulti-epitopic, peptide can then be generated synthetically,recombinantly, or via cleavage from the native source.

The peptides of the invention can be prepared in a wide variety of ways.For the preferred relatively short size, the peptides can be synthesizedin solution or on a solid support in accordance with conventionaltechniques. Various automatic synthesizers are commercially availableand can be used in accordance with known protocols. See, for example,Stewart & Young, SOLID PHASE PEPTIDE SYNTHESIS, 2D. ED., Pierce ChemicalCo. (1984). Further, individual peptides may be joined using chemicalligation to produce larger peptides.

Alternatively, recombinant DNA technology may be employed wherein anucleotide sequence which encodes an immunogenic peptide of interest isinserted into an expression vector, transformed or transfected into anappropriate host cell and cultivated under conditions suitable forexpression. These procedures are generally known in the art, asdescribed generally in Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989). Thus,recombinant polypeptides which comprise one or more peptide sequences ofthe invention can be used to present the appropriate T cell epitope.

As the nucleotide coding sequence for peptides of the preferred lengthscontemplated herein can be synthesized by chemical techniques, forexample, the phosphotriester method of Matteucci, et al., J. Am. Chem.Soc. 103:3185 (1981) modification can be made simply by substituting theappropriate and desired nucleic acid base(s) for those that encode thenative peptide sequence. The coding sequence can then be provided withappropriate linkers and ligated into expression vectors commonlyavailable in the art, and the vectors used to transform suitable hoststo produce the desired fusion protein. A number of such vectors andsuitable host systems are now available. For expression of the fusionproteins, the coding sequence will be provided with operably linkedstart and stop codons, promoter and terminator regions and usually areplication system to provide an expression vector for expression in thedesired cellular host. For example, promoter sequences compatible withbacterial hosts are provided in plasmids containing convenientrestriction sites for insertion of the desired coding sequence. Theresulting expression vectors are transformed into suitable bacterialhosts. Of course, yeast, insect or mammalian cell hosts may also beused, employing suitable vectors and control sequences.

IV.H. Peptide Epitope Reagents to Evaluate Immune Responses.

HLA class I and class II binding peptides as described herein can beused, in one embodiment of the invention, as reagents to evaluate animmune response. The immune response to be evaluated may be induced byusing as an immunogen any agent that would potentially result in theproduction of antigen-specific CTLs or HTLs to the peptide epitope(s) tobe employed as the reagent. The peptide reagent is not used as theimmunogen.

For example, a peptide of the invention may be used in a tetramerstaining assay to assess peripheral blood mononuclear cells for thepresence of antigen-specific CTLs following exposure to a pathogen orimmunogen. The HLA-tetrameric complex is used to directly visualizeantigen-specific CTLs (see, e.g., Ogg et al. Science 279:2103-2106,1998; and Altman et al. Science 174:94-96, 1996) and determine thefrequency of the antigen-specific CTL population in a sample ofperipheral blood mononuclear cells. A tetramer reagent using a peptideof the invention may be generated as follows: A peptide that binds to anallele-specific HLA molecules, or supertype molecules, is refolded inthe presence of the corresponding HLA heavy chain and β₂-microglobulinto generate a trimolecular complex. The complex is biotinylated at thecarboxyl terminal end of the heavy chain at a site that was previouslyengineered into the protein. Tetramer formation is then induced by theaddition of streptavidin. By means of fluorescently labeledstreptavidin, the tetramer can be used to stain antigen-specific cells.The cells may then be identified, for example, by flow cytometry. Suchan analysis may be used for diagnostic or prognostic purposes.

Peptides of the invention may also be used as reagents to evaluateimmune recall responses. (see, e.g., Bertoni et al. J. Clin. Invest.100:503-513, 1997 and Penna et al. J. Exp. Med. 174:1565-1570, 1991.)For example, patient PBC samples from individuals with acute hepatitis Bor who have recently recovered from acute hepatitis B may be analyzedfor the presence of HBV antigen-specific CTLs using HBV-specificpeptides. A blood sample containing mononuclear cells may be evaluatedby cultivating the PBCs and stimulating the cells with a peptide of theinvention. After an appropriate cultivation period, the expanded cellpopulation may be analyzed for cytotoxic activity.

The peptides may also be used as reagents to evaluate the efficacy of avaccine. PBMCs obtained from a patient vaccinated with an immunogen maybe analyzed using, for example, either of the methods described above. Apatient is HLA typed, and appropriate peptide reagents that recognizeallele-specific molecules present in that patient may be selected forthe analysis. The immunogenicity of the vaccine will be indicated by thepresence of HBV epitope-specific CTLs in the PBMC sample.

IV.I. Vaccine Compositions

Vaccines that contain as an active ingredient an immunogenicallyeffective amount of one or more peptides as described herein are afurther embodiment of the invention. Once appropriately immunogenicepitopes have been defined, they can be sorted and delivered by variousmeans, herein referred to as “vaccine” compositions. Such vaccinecompositions can include, for example, lipopeptides (Vitiello, A. etal., J. Clin. Invest. 95:341, 1995), peptides compositions encapsulatedin poly(DL-lactide-co-glycolide) (PLG) microspheres (see, e.g.,Eldridge, et al. Molec. Immunol. 28:287-294, 1991: Alonso et al. Vaccine12:299-306, 1994; Jones et al. Vaccine 13:675-681, 1995), peptidecompositions encapsulated in immune stimulating complexes (ISCOMS) (see,e.g., Takahashi et al. Nature 344:873-875, 1990; Hu et al. Clin ExpImmunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs)(see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988;Tam, J. P., J. Immunol. Methods 196:17-32, 1996), viral delivery vectors(Perkus, M. E. et al., In: Concepts in vaccine development, Kaufmann, S.H. E., ed., p. 379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986;Hu, S. L. et al., Nature 320:537, 1986; Kieny, M. -P. et al., AIDSBio/Technology 4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148,1971; Chanda, P. K. et al., Virology 175:535, 1990), particles of viralor synthetic origin (Kofler, N. et al., J. Immunol. Methods. 192:25,1996; Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D.,Jr. et al., Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel,F. R., and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K.et al., Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol.148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked orparticle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993;Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993;Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S.H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev.Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16,1993). Toxin-targeted, also know as receptor mediated targeting,delivery technologies also may be used such as those of AvantImmunotherapeutics, Inc. (Needham, Mass.).

Furthermore, vaccines in accordance with the invention encompasscompositions of one or more of the claimed peptide(s) that can beintroduced into a host, including humans, linked to its own carrier, oras a homopolymer or heteropolymer of active peptide units., Such apolymer has the advantage of increased immunological reaction and, wheredifferent peptides are used to make up the polymer, the additionalability to induce antibodies and/or CTLs that react with differentantigenic determinants of the pathogenic organism or tumor-relatedpeptide targetted for an immune response.

Furthermore, useful carriers that can be used with vaccines of theinvention are well known in the art, and include, e.g., thyroglobulin,albumins such as human serum albumin, tetanus toxoid, polyamino acidssuch as poly L-lysine, poly L-glutamic acid, influenza, hepatitis Bvirus core protein, hepatitis B virus recombinant vaccine and the like.The vaccines can contain a physiologically tolerable (i.e., acceptable)diluent such as water, or saline, preferably phosphate buffered saline.The vaccines also typically include an adjuvant. Adjuvants such asincomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, oralum are examples of materials well known in the art. Additionally, asdisclosed herein, CTL responses can be primed by conjugating peptides ofthe invention to lipids, such astripalmitoyl-S-glycerylcysteinlyseryl-serine P₃CSS).

As disclosed in greater detail herein, upon immunization with a peptidecomposition in accordance with the invention, via injection, aerosol,oral, transdermal, transmucosal, intrapleural, intrathecal, or othersuitable routes, the immune system of the host responds to the vaccineby producing large amounts of CTLs specific for the desired antigen, andthe host becomes at least partially immune to later infection, or atleast partially resistant to developing an ongoing chronic infection.

In some instances it may be desirable to combine the class I peptidevaccines of the invention with vaccines which induce or facilitateneutralizing antibody responses to the target antigen of interest,particularly to viral envelope antigens. A preferred embodiment of sucha composition comprises class I and class II epitopes in accordance withthe invention. An alternative embodiment of such a composition comprisesa class I and/or class II epitope in accordance with the invention,along with a PADRE™ (Epimmune, San Diego, Calif.) molecule (described inthe related U.S. Ser. No. 08/485,218, which is a CIP of U.S. Ser. No.08/305,871, now U.S. Pat. No. 5,736,142, which is a CIP of abandonedapplication U.S. Ser. No. 08/121,101.) Furthermore, any of theseembodiments can be administered as a nucleic acid mediated modality.

For therapeutic or immunization purposes, the peptides of the inventioncan also be expressed by viral or bacterial vectors. Examples ofexpression vectors include attenuated viral hosts, such as vaccinia orfowlpox. This approach involves the use of vaccinia virus as a vector toexpress nucleotide sequences that encode the peptides of the invention.Upon introduction into an acutely or chronically infected host or into anon-infected host, the recombinant vaccinia virus expresses theimmunogenic peptide, and thereby elicits a host CTL and/or HTL response.Vaccinia vectors and methods useful in immunization protocols aredescribed in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG(Bacille Calmette Guerin). BCG vectors are described in Stover, et al.Nature 351:456-460 (1991). A wide variety of other vectors useful fortherapeutic administration or immunization of the peptides of theinvention, e.g. adeno and adeno-associated virus vectors, retroviralvectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, andthe like, will be apparent to those skilled in the art from thedescription herein.

Antigenic peptides are used to elicit a CTL and/or HTL response ex vivo,as well. The resulting CTL or HTL cells, can be used to treat chronicinfections, or tumors in patients that do not respond to otherconventional forms of therapy, or will not respond to a therapeuticvaccine peptide or nucleic acid in accordance with the invention. Exvivo CTL or HTL responses to a particular pathogen (infectious agent ortumor antigen) are induced by incubating in tissue culture the patient'sCTL or HTL precursor cells together with a source of antigen-presentingcells (APC), such as dendritic cells, and the appropriate immunogenicpeptide. After an appropriate incubation time (typically about 14weeks), in which the precursor cells are activated, mature and expandinto effector cells, the cells are infused back into the patient, wherethey will destroy (CTL) or facilitate destruction (HTL) of theirspecific target cell (an infected cell or a tumor cell).

Transfected dendritic cells may also be used as antigen presentingcells. Alternatively, dendritic cells are transfected, e.g., with aminigene construct in accordance with the invention, in order to elicitimmune responses. Minigenes will be discussed in greater detail in afollowing section.

DNA or RNA encoding one or more of the peptides of the invention canalso be administered to a patient. This approach is described, forinstance, in Wolff et. al., Science 247:1465 (1990) as well as U.S. Pat.Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647;WO 98/04720; and in more detail below. Examples of DNA-based deliverytechnologies include “naked DNA”, facilitated (bupivicaine, polymers,peptide-mediated) delivery, cationic lipid complexes, andparticle-mediated (“gene gun”) delivery.

Preferably, the following principles are utilized when selecting anarray of epitopes for inclusion in a polyepitopic composition, or forselecting epitopes to be included in a vaccine composition and/or to beencoded by a minigene. It is preferred that each of the followingprinciples are balanced in order to make the selection.

1.) Epitopes are selected which, upon administration, mimic immuneresponses that have been observed to be correlated with HBV clearance.For HLA Class I this includes 3-4 epitopes that come from at least oneantigen of HBV. In other words, it has been observed that in patientswho spontaneously clear HBV, that they had generated an immune responseto at least 3 epitopes on at least one HBV antigen. For HLA Class II asimilar rationale is employed; again 3-4 epitopes are selected from atleast one HBV antigen (see e.g., Rosenberg et al. Science278:1447-1450).

2.) Epitopes are selected that have the requisite binding affinityestablished to be correlated with immunogenicity: for HLA Class I anIC₅₀ of 500 nM or less, or for Class II an IC₅₀ of 1000 nM or less.

3.) Sufficient supermotif bearing peptides, or a sufficient array ofallele-specific motif bearing peptides, are selected to give broadpopulation coverage. For example, it is preferable to have at least 80%population coverage. A Monte Carlo analysis, a statistical evaluationknown in the art, can be employed to assess population coverage.

4.) When selecting epitopes from cancer-related antigens it is oftenpreferred to select analogs. When selecting epitopes for infectiousdisease-related antigens it is often preferable to select nativeepitopes. Therefore, of particular relevance for infectious diseasevaccines (but for cancer-related vaccines as well), are epitopesreferred to as “nested epitopes.” Nested epitopes occur where at leasttwo epitopes overlap in a given peptide sequence. A peptide comprising“transcendent nested epitopes” is a peptide that has both HLA class Iand HLA class II epitopes in it.

When providing nested epitopes, it is preferable to provide a sequencethat has the greatest number of epitopes per provided sequence. Alimitation on this principle is to avoid providing a peptide that is anylonger than the amino terminus of the amino terminal epitope and thecarboxyl terminus of the carboxyl terminal epitope in the peptide. Whenproviding a longer peptide sequence, such as a sequence comprisingnested epitopes, it is important to screen the sequence in order toinsure that it does not have pathological or other deleteriousbiological properties.

5.) When creating a minigene, as disclosed in greater detail in thefollowing section, an objective is to generate the smallest peptidepossible that encompasses the epitopes of interest. The principlesemployed are similar, if not the same as those employed when selecting apeptide comprising nested epitopes. Thus, upon determination of thenucleic acid sequence to be provided as a minigene, the peptide encodedthereby is analyzed to determine whether any “junctional epitopes” havebeen created. A junctional epitope is an actual binding epitope, aspredicted, e.g., by motif analysis. Junctional epitopes are to beavoided because the recipient may generate an immune response to thatepitope. Of particular concern is a junctional epitope that is a“dominant epitope.” A dominant epitope may lead to such a zealousresponse that immune responses to other epitopes are diminished orsuppressed.

IV.I.1. Minigene Vaccines

A growing body of experimental evidence demonstrates that a number ofdifferent approaches are available which allow simultaneous delivery ofmultiple epitopes. Nucleic acids encoding the peptides of the inventionare a particularly useful embodiment of the invention. Epitopes forinclusion in a minigene are preferably selected according to theguidelines above. A preferred means of administering nucleic acidsencoding the peptides of the invention uses minigene constructs encodingone or multiple epitopes of the invention. The use of multi-epitopeminigenes is described below and in, e.g. An, L. and Whitton, J. L., J.Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822, 1996;Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine16:426, 1998. For example, a multi-epitope DNA plasmid encoding ninedominant HLA-A*0201- and A11-restricted epitopes derived from thepolymerase, envelope, and core proteins of HBV and HIV, the PADRE™universal helper T cell (HTL) epitope, and an ER-translocating signalsequence was engineered. Immunization of HLA transgenic mice with thisplasmid construct resulted in strong CTL induction responses against thenine epitopes tested, similar to those observed with a lipopeptide ofknown immunogenicity in humans, and significantly greater thanimmunization in oil-based adjuvants. Moreover, the immunogenicity ofDNA-encoded epitopes in vivo correlated with the in vitro responses ofspecific CTL lines against target cells transfected with the DNAplasmid.

For example, to create a DNA sequence encoding the selected epitopes(minigene) for expression in human cells, the amino acid sequences ofthe epitopes may be reverse translated. A human codon usage table can beused to guide the codon choice for each amino acid. Theseepitope-encoding DNA sequences may be directly adjoined, so that whentranslated, a continuous polypeptide sequence is created. To optimizeexpression and/or immunogenicity, additional elements can beincorporated into the minigene design. Examples of amino acid sequencesthat could be reverse translated and included in the minigene sequenceinclude: HLA class I epitopes, HLA class II epitopes, a ubiquitinationsignal sequence, a leader sequence, and/or an endoplasmic reticulumtargeting signal. In addition, HLA presentation of CTL and HTL epitopesmay be improved by including synthetic (e.g. poly-alanine) ornaturally-occurring flanking sequences adjacent to the CTL or HTLepitopes.

The minigene sequence may be converted to DNA by assemblingoligonucleotides that encode the plus and minus strands of the minigene.Overlapping oligonucleotides (30-100 bases long) may be synthesized,phosphorylated, purified and annealed under appropriate conditions usingwell known techniques. The ends of the oligonucleotides can be joined,for example, using T4 DNA ligase. This synthetic minigene, encoding theepitope polypeptide, can then be cloned into a desired expressionvector.

Standard regulatory sequences well known to those of skill in the artare preferably included in the vector to ensure expression in the targetcells. Several vector elements are desirable: a promoter with adown-stream cloning site for minigene insertion; a polyadenylationsignal for efficient transcription termination; an E. coli origin ofreplication; and an E. coli selectable marker (e.g. ampicillin orkanamycin resistance). Numerous promoters can be used for this purpose,e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat.Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

Additional vector modifications may be desired to optimize minigeneexpression and immunogenicity. In some cases, introns are required forefficient gene expression, and one or more synthetic ornaturally-occurring introns could be incorporated into the transcribedregion of the minigene. The inclusion of mRNA stabilization sequencesand sequences for replication in mammalian cells may also be consideredfor increasing minigene expression.

Once an expression vector is selected, the minigene is cloned into thepolylinker region downstream of the promoter. This plasmid istransformed into an appropriate E. coli strain, and DNA is preparedusing standard techniques. The orientation and DNA sequence of theminigene, as well as all other elements included in the vector, areconfirmed using restriction mapping and DNA sequence analysis. Bacterialcells harboring the correct plasmid can be stored as a master cell bankand a working cell bank.

In addition, immunostimulatory sequences (ISSs or CpGs) appear to play arole in the immunogenicity of DNA vaccines. These sequences may beincluded in the vector, outside the minigene coding sequence, if desiredto enhance immunogenicity.

In some embodiments, a bi-cistronic expression vector which allowsproduction of both the minigene-encoded epitopes and a second protein(included to enhance or decrease immunogenicity) can be used. Examplesof proteins or polypeptides that could beneficially enhance the immuneresponse if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF),cytokine-inducing molecules (e.g., LeIF) or costimulatory molecules.Helper (HTL) epitopes can be joined to intracellular targeting signalsand expressed separately from expressed CTL epitopes; this allowsdirection of the HTL epitopes to a cell compartment different than thatof the CTL epitopes. If required, this could facilitate more efficiententry of HTL epitopes into the HLA class II pathway, thereby improvingCTL induction. In contrast to HTL or CTL induction, specificallydecreasing the immune response by co-expression of immunosuppressivemolecules (e.g. TGF-β) may be beneficial in certain diseases).

Therapeutic quantities of plasmid DNA can be produced for example, byfermentation in E. coli, followed by purification. Aliquots from theworking cell bank are used to inoculate growth medium, and grown tosaturation in shaker flasks or a bioreactor according to well knowntechniques. Plasmid DNA can be purified using standard bioseparationtechnologies such as solid phase anion-exchange resins supplied byQIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can beisolated from the open circular and linear forms using gelelectrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using a variety offormulations. The simplest of these is reconstitution of lyophilized DNAin sterile phosphate-buffer saline (PBS). This approach, known as “nakedDNA,” is currently being used for intramuscular (IM) administration inclinical trials. To maximize the immunotherapeutic effects of minigeneDNA vaccines, an alternative method for formulating purified plasmid DNAmay be desirable. A variety of methods have been described, and newtechniques may become available. Cationic lipids can also be used in theformulation (see, e.g., as described by WO 93/24640; Mannino &Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S. Pat. No. 5,279,833;WO 91/06309; and Felgner, et al., Proc. Nat'l Acad. Sci. USA 84:7413(1987). In addition, glycolipids, fusogenic liposomes, peptides andcompounds referred to collectively as protective, interactive,non-condensing (PINC) could also be complexed to purified plasmid DNA toinfluence variables such as stability, intramuscular dispersion, ortrafficking to specific organs or cell types.

Target cell sensitization can be used as a functional assay forexpression and HLA class I presentation of minigene-encoded CTLepitopes, respectively. For example, the plasmid DNA is introduced intoa mammalian cell line that is suitable as a target for standard CTLchromium release assays. The transfection method used will be dependenton the final formulation. Electroporation can be used for “naked” DNA,whereas cationic lipids allow direct in vitro transfection. A plasmidexpressing green fluorescent protein (GFP) can be co-transfected toallow enrichment of transfected cells using fluorescence activated cellsorting (FACS). These cells are then chromium-51 (⁵¹Cr) labeled and usedas target cells for epitope-specific CTL lines; cytolysis, detected by⁵¹Cr release, indicates production of HLA presentation ofminigene-encoded CTL epitopes.

In vivo immunogenicity is a second approach for functional testing ofminigene DNA formulations. Transgenic mice expressing appropriate humanHLA proteins are immunized with the DNA product. The dose and route ofadministration are formulation dependent (e.g., IM for DNA in PBS, IPfor lipid-complexed DNA). Twenty-one days after immunization,splenocytes are harvested and restimulated for 1 week in the presence ofpeptides encoding each epitope being tested. For CTL effector cells,assays are conducted for cytolysis of peptide-loaded, chromium-51labeled target cells using standard techniques. Lysis of target cellssensitized by HLA loading of peptides corresponding to minigene-encodedepitopes demonstrates DNA vaccine function for in vivo induction ofCTLs.

Alternatively, the nucleic acids can be administered using ballisticdelivery as described, for instance, in U.S. Pat. No. 5,204,253. Usingthis technique, particles comprised solely of DNA are administered. In afurther alternative embodiment, DNA can be adhered to particles, such asgold particles.

IV.I.2. Combinations with Helper Pepides

The peptides of the present invention, or analogs thereof, which haveimmunostimulatory activity may be modified to provide desiredattributes, such as improved serum half life, or to enhanceimmunogenicity.

For instance, the ability of the peptides to induce CTL activity can beenhanced by linking the peptide to a sequence which contains at leastone epitope that is capable of inducing a T helper cell response.Particularly preferred immunogenic peptides/T helper conjugates arelinked by a spacer molecule. The spacer is typically comprised ofrelatively small, neutral molecules, such as amino acids or amino acidmimetics, which are substantially uncharged under physiologicalconditions. The spacers are typically selected from, e.g., Ala, Gly, orother neutral spacers of nonpolar amino acids or neutral polar aminoacids. It will be understood that the optionally present spacer need notbe comprised of the same residues and thus may be a hetero- orhomo-oligomer. When present, the spacer will usually be at least one ortwo residues, more usually three to six residues. Alternatively, the CTLpeptide may be linked to the T helper peptide without a spacer.

The immunogenic peptide may be linked to the T helper peptide eitherdirectly or via a spacer either at the amino or carboxy terminus of theCTL peptide. The amino terminus of either the immunogenic peptide or theT helper peptide may be acylated. The T helper peptides used in theinvention can be modified in the same manner as CTL peptides. Forinstance, they may be modified to include D-amino acids or be conjugatedto other molecules such as lipids, proteins, sugars and the like.Exemplary T helper peptides include tetanus toxoid 830-843, influenza307-319, and malarial circumsporozoite 382-398 and 378-389.

In certain embodiments, the T helper peptide is one that is recognizedby T helper cells present in the majority of the population. This can beaccomplished by selecting amino acid sequences that bind to many, most,or all of the HLA class II molecules. These are known as “looselyHLA-restricted” or “promiscuous” T helper sequences. Examples of aminoacid sequences that are promiscuous include sequences from antigens suchas tetanus toxoid at positions 830-843 (QYIKANSKFIGITE), Plasmodiumfalciparum CS protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS), andStreptococcus 18 kD protein at positions 116 (GAVDSILGGVATYGAA). Otherexamples include peptides bearing a DR 1-4-7 supermotif.

Alternatively, it is possible to prepare synthetic peptides capable ofstimulating T helper lymphocytes, in a loosely HLA-restricted fashion,using amino acid sequences not found in nature (see, e.g., PCTpublication WO 95/07707). These synthetic compounds calledPan-DR-binding epitopes (e.g., PADRE™, Epimmune, Inc., San Diego,Calif.) are designed on the basis of their binding activity to mostHLA-DR (human HLA class II) molecules. For instance, a pan-DR-bindingepitope peptide having the formula: aKXVWANTLKAAa, where “X” is eithercyclohexylalanine, phenylalanine, or tyrosine, and a is either D-alanineor L-alanine, has been found to bind to most HLA-DR alleles, and tostimulate the response of T helper lymphocytes from most individuals,regardless of their HLA type.

T helper epitopes can also be modified to alter their biologicalproperties. For example, peptides presenting T helper epitopes cancontain D-amino acids to increase their resistance to proteases and thusextend their serum half-life. Also, the epitope peptides of theinvention can be conjugated to other molecules such as lipids, proteinsor sugars, or any other synthetic compounds, to increase theirbiological activity. Specifically, the T helper peptide can beconjugated to one or more palmitic acid chains at either the amino orcarboxyl termini.

In some embodiments it may be desirable to include in the pharmaceuticalcompositions of the invention at least one component which primescytotoxic T lymphocytes. Lipids have been identified as agents capableof priming CTL in vivo against viral antigens. For example, palmiticacid residues can be attached to the ε-and α-amino groups of a lysineresidue and then linked, e.g., via one or more linking residues such asGly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. Thelipidated peptide can then be administered either directly in a micelleor particle, incorporated into a liposome, or emulsified in an adjuvant,e.g., incomplete Freund's adjuvant. In a preferred embodiment, aparticularly effective immunogenic comprises palmitic acid attached toε- and α-amino groups of Lys, which is attached via linkage, e.g.,Ser-Ser, to the amino terminus of the immunogenic peptide.

As another example of lipid priming of CTL responses, E. colilipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine(P₃CSS) can be used to prime virus specific CTL when covalently attachedto an appropriate peptide. See, Deres, et al., Nature 342:561 (1989).Peptides of the invention can be coupled to P₃CSS, for example, and thelipopeptide administered to an individual to specifically prime a CTLresponse to the target antigen. Moreover, because the induction ofneutralizing antibodies can also be primed with P₃CSS-conjugatedepitopes, two such compositions can be combined to more effectivelyelicit both humoral and cell-mediated responses to infection.

In addition, additional amino acids can be added to the termini of apeptide to provide for ease of linking peptides one to another, forcoupling to a carrier support, or larger peptide, for modifying thephysical or chemical properties of the peptide or oligopeptide, or thelike. Amino acids such as tyrosine, cysteine, lysine, glutamic oraspartic acid, or the like, can be introduced at the C- or N-terminus ofthe peptide or oligopeptide, particularly class I peptides. However, itis to be noted that modification at the carboxyl terminus may, in somecases, alter binding characteristics of the peptide. In addition, thepeptide or oligopeptide sequences can differ from the natural sequenceby being modified by terminal-NH₂ acylation, e.g., by alkanoyl (C₁-C₂₀)or thioglycolyl acetylation, terminal-carboxylamidation, e.g., ammonia,methylamine, etc. In some instances these modifications may providesites for linking to a support or other molecule.

IV.J. Administration of Vaccines for Therapeutic or ProphylacticPurposes

The peptides of the present invention and pharmaceutical and vaccinecompositions of the invention are useful for administration to mammals,particularly humans, to treat and/or prevent HBV infection. Vaccinecompositions containing the peptides of the invention are administeredto a patient susceptible to or otherwise at risk for HBV infection toelicit an immune response against HBV antigens and thus enhance thepatient's own immune response capabilities. In therapeutic applications,compositions are administered to a patient in an amount sufficient toelicit an effective CTL response to the virus or tumor antigen and tocure or at least partially arrest or slow symptoms and/or complications.An amount adequate to accomplish this is defined as “therapeuticallyeffective dose.” Amounts effective for this use will depend on, e.g.,the particular composition administered, the manner of administration,the stage and severity of the disease being treated, the weight andgeneral state of health of the patient, and the judgment of theprescribing physician. Generally the dosage range for an initialimmunization (i.e., therapeutic or prophylactic administration) isbetween about 1.0 μg to about 5000 μg of peptide, typically betweenabout 10 μg to about 1000 μg, for a 70 kg patient, followed by boostingdosages of between about 1.0 μg to about 5000 μg of peptide pursuant toa boosting regimen over weeks to months depending upon the patient'sresponse and condition as determined by measuring specific CTL activityin the patient's blood. The peptides and compositions of the presentinvention may be employed in serious disease states, that is,life-threatening or potentially life threatening situations. In suchcases, as a result of the minimal amounts of extraneous substances andthe relative nontoxic nature of the peptides in preferred compositionsof the invention, it is possible and may be felt desirable by thetreating physician to administer substantial excesses of these peptidecompositions relative to these stated dosage amounts.

As noted above, the “CTL” peptides of the invention induce immuneresponses when contacted with a CTL specific to an epitope comprised bythe peptide. The manner in which the peptide is contacted with the CTLis not critical to the invention. For instance, the peptide can becontacted with the CTL either in vivo or in vitro. If the contactingoccurs in vivo, the peptide itself can be administered to the patient,or other vehicles, e.g., DNA vectors encoding one or more peptides,vital vectors encoding the peptide(s), liposomes and the like, can beused, as described herein.

For pharmaceutical compositions, the immunogenic peptides, or DNAencoding them, are generally administered to an individual alreadyinfected with HBV. The peptides or DNA encoding them can be administeredindividually or as fusions of one or more peptide sequences. Those inthe incubation phase or the acute phase of infection can be treated withthe immunogenic peptides separately or in conjunction with othertreatments, as appropriate.

For therapeutic use, administration should generally begin at the firstdiagnosis of HBV infection. This is followed by boosting doses until atleast symptoms are substantially abated and for a period thereafter. Inchronic infection, loading doses followed by boosting doses may berequired.

Treatment of an infected individual with the compositions of theinvention may hasten resolution of the infection in acutely infectedindividuals. For those individuals susceptible (or predisposed) todeveloping chronic infection, the compositions are particularly usefulin methods for preventing the evolution from acute to chronic infection.Where susceptible individuals are identified prior to or duringinfection, the composition can be targeted to them, minimizing need foradministration to a larger population.

The peptide or other compositions as used for the treatment of chronicHBV infection and to stimulate the immune system to eliminatepathogen-infected cells in, e.g., persons who have not manifestedsymptoms of disease but who act as a disease vector. In this context, itis generally important to provide an amount of immuno-potentiatingpeptide in a formulation and mode of administration sufficient toeffectively stimulate a cytotoxic T cell response; compositions whichstimulate helper T cell responses can also be given in accordance withthis embodiment of the invention. Thus, for treatment of chronicinfection, a representative dose is in the range of about 1.0 μg toabout 5000 μg, preferably about 10 μg to 1000 μg, per 70 kg patientweight per dose. Immunizing doses followed by boosting doses atestablished intervals, e.g., from four weeks to six months, may berequired, possibly for a prolonged period of time to effectivelyimmunize an individual. In the case of chronic infection, administrationshould continue until at least clinical symptoms or laboratory testsindicate that the viral infection has been eliminated or substantiallyabated and for a period thereafter. The dosages, routes ofadministration, and dose schedules are adjusted in accordance withmethodologies known in the art.

The pharmaceutical compositions for therapeutic treatment are intendedfor parenteral, topical, oral, intrathecal, or local administration.Preferably, the pharmaceutical compositions are administered parentally,e.g., intravenously, subcutaneously, intradermally, or intramuscularly.Thus, the invention provides compositions for parenteral administrationwhich comprise a solution of the immunogenic peptides dissolved orsuspended in an acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers may be used, e.g., water, buffered water,0.8% saline, 0.3% glycine, hyaluronic acid and the like. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH-adjusting and buffering agents, tonicityadjusting agents, wetting agents, preservatives, and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc.

The concentration of peptides of the invention in the pharmaceuticalformulations can vary widely, ie., from less than about 0.1%, usually ator at least about 2% to as much as 20% to 50% or more by weight, andwill be selected primarily by fluid volumes, viscosities, etc., inaccordance with the particular mode of administration selected.

The peptides of the invention may also be administered via liposomes,which serve to target the peptides to a particular tissue, such aslymphoid tissue, or targeted selectively to infected cells, as well asincrease the half-life of the peptide composition. Liposomes includeemulsions, foams, micelles, insoluble monolayers, liquid crystals,phospholipid dispersions, lamellar layers and the like. In thesepreparations the peptide to be delivered is incorporated as part of aliposome, alone or in conjunction with a molecule which binds to, e.g.,a receptor prevalent among lymphoid cells, such as monoclonal antibodieswhich bind to the CD45 antigen, or with other therapeutic or immunogeniccompositions. Thus, liposomes either filled or decorated with a desiredpeptide of the invention can be directed to the site of lymphoid cells,where the liposomes then deliver the peptide compositions. Liposomes foruse in the invention are formed from standard vesicle-forming lipids,which generally include neutral and negatively charged phospholipids anda sterol, such as cholesterol. The selection of lipids is generallyguided by consideration of, e.g., liposome size, acid lability andstability of the liposomes in the blood stream. A variety of methods areavailable for preparing liposomes, as described in, e.g., Szoka, et al.,Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871,4,501,728,4,837,028, and 5,019,369.

For targeting cells of the immune system, a ligand to be incorporatedinto the liposome can include, e.g., antibodies or fragments thereofspecific for cell surface determinants of the desired immune systemcells. A liposome suspension containing a peptide may be administeredintravenously, locally, topically, etc. in a dose which varies accordingto, inter alia, the manner of administration, the peptide beingdelivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, one or more peptides of the invention, and morepreferably at a concentration of 25%-75%.

For aerosol administration, the immunogenic peptides are preferablysupplied in finely divided form along with a surfactant and propellant.Typical percentages of peptides are 0.01%-20% by weight, preferably1%-10%. The surfactant must, of course, be nontoxic, and preferablysoluble in the propellant. Representative of such agents are the estersor partial esters of fatty acids containing from 6 to 22 carbon atoms,such as caproic, octanoic, lauric, palmitic, stearic, linoleic,linolenic, olesteric and oleic acids with an aliphatic polyhydricalcohol or its cyclic anhydride. Mixed esters, such as mixed or naturalglycerides may be employed. The surfactant may constitute 0.1%-20% byweight of the composition, preferably 0.25-5%. The balance of thecomposition is ordinarily propellant. A carrier can also be included, asdesired, as with, e.g., lecithin for intranasal delivery.

The vaccine compositions of the invention may also be used purely asprophylactic agents. Vaccine compositions containing the peptideepitopes of the invention are administered to a patient susceptible to,or otherwise at risk for, HBV infection to elicit an immune responseagainst HBV antigens and thus enhance the patient's own immune responsecapabilities following exposure to HBV. Generally the dosage range foran initial prophylactic immunization is between about 1.0 μg to about5000 μg of peptide, typically between about 10 μg to about 1000 μg, fora 70 kg patient. This is followed by boosting dosages of between about1.0 μg to about 5000 μg of peptide administered at defined intervalsfrom about four weeks to six months after the initial administration ofvaccine. The immunogenicity of the vaccine may be assessed by measuringspecific CTL activity in the patient's blood.

IV.K. Kits

The peptide and nucleic acid compositions of this invention can beprovided in kit form together with instructions for vaccineadministration. Typically the kit would include desired peptidecompositions in a container, preferably in unit dosage form andinstructions for administration. An alternative kit would include aminigene construct with desired nucleic acids of the invention in acontainer, preferably in unit dosage form together with instruction foradministration. Lymphokines such as IL-2 or IL-12 may also be includedin the kit. Other kit components that may also be desirable include, forexample, a sterile syringe, booster dosages, and other desiredexcipients.

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner. Those ofskill in the art will readily recognize a variety of non-criticalparameters that can be changed or modified to yield alternativeembodiments in accordance with the invention.

V. EXAMPLES Example 1 HLA Class I Binding Assays

The following example of peptide binding to HLA-A3 supertype moleculesdemonstrates quantification of binding affinities of HLA class Ipeptides. Analogous binding assays can be performed for other peptidesthat bind class I or class II HLA molecules. Furthermore, binding assayscan be performed with peptides that are not motif-bearing.

For example, the affinity of peptides bearing an HLA-A3 supermotif wasdetermined as follows. Epstein-Barr virus (EBV)-transformed homozygouscell lines were used as sources of class I molecules. Cell linesinclude, e.g., GM3107 (A3, B7; Human Genetic Mutant Repository); BVR(A11, B35.3, Cw4; Human Genetic Mutant Repository); SPACH (A31, B62,Cw1/3; ASHI Repository Collection); LWAGS (A*3301, B14, and Cw8; ASHIRepository Collection) (Bodmer, et al., Hum. Immunol. 43:149, 1995), anda C1R transfectant characterized by Dr. Walter Storkus (University ofPittsburgh) for the isolation of A*6801. Cell lines were maintained aspreviously described (Sidney, et al., J. Immunol. 154:247 (1995); Sette,et al., Mol. Immunol. 31:813 (1994)).

Cell lysates were prepared and HLA class I molecules purified inaccordance with disclosed protocols (Sidney, et al., J. Immunol. 154:247(1995); Sette, et al., Mol. Immunol. 31:813 (1994)). Briefly, cells werelysed at a concentration of 10⁸ cells/ml in 50 mM Tris-HCl, pH 8.5,containing 1% Nonidet P-40 (Fluka Biochemika, Buchs, Switzerland), 150mM NaCl, 5 mM EDTA, and 2 mM PMSF. The lysates were passed through 0.45μM filters and cleared of nuclei and debris by centrifugation at 10,000g for 20 minutes. HILA proteins were then purified by affinitychromatography. Columns of inactivated Sepharose CL 4B and Protein ASepharose were used as precolumns. The cell lysate was depleted of HLA-Band HLA-C proteins by repeated passage over Protein A Sepharose beadsconjugated with the anti-HLA(B,C) antibody B1.23.2 (Rebai, et al.,Tissue Antigens 22:107 (1983)). Typically two to four passages wererequired for effective depletion. Subsequently, the anti HLA(A,B,C)antibody W6/32 (Barnstable, et al., Cell 14:9 (1978)) was used tocapture HLA-A molecules. Protein purity, concentration, andeffectiveness of depletion steps were monitored by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Binding Assays

Quantitative assays for the binding of peptides to soluble class Imolecules on the basis of the inhibition of binding of a radiolabeledstandard probe peptide to detergent solubilized HLA molecules wereperformed as described in the literature (Kubo, et al., J. Immunol.152:3913 (1994); Kast, et al., J. Immunol. 152:3904 (1994); Sidney, etal., J. Immunol. 154:247 (1995); Sette, et al., Mol. Immunol. 31:813(1994); Ruppert, et al., Cell 74:929 (1993)). Briefly, 1-10 nM ofradiolabeled probe peptide, iodinated by the Chloramine-T method(Greenwood, et al., Biochem. J. 89:114 (1963)), was co-incubated at roomtemperature with various amounts of HLA in the presence of 1 μM humanβ₂-microglobulin (Scripps Laboratories, San Diego, Calif., USA) and acocktail of protease inhibitors. At the end of a two day incubationperiod, the percent of HLA-bound radioactivity was determined by sizeexclusion gel filtration chromatography on a TSK 2000 column.

The A3CON1 peptide (sequence KVFPYALINK) (Kubo, et al., J. Immunol.152:3913 (1994)) was used as the radiolabeled probe for the A3, A11,A31, and A*6801 assays. A T7Y analogue of HBVc 141-151 (sequenceSTLPETYVVRR) (Missale, et al., J. Exp. Med. 177:751 (1993)) was used asthe radiolabeled probe for the A*3301 assay. In the case of competitiveassays, the concentration of peptide yielding 50% inhibition of thebinding of the radiolabeled probe peptide (IC₅₀) was calculated.Peptides were usually tested at one or two high doses, and the IC₅₀ ofpeptides yielding positive inhibition were determined in subsequentexperiments, in which two to six further dilutions were tested, asnecessary. To achieve a suitable signal, HLA concentrations yieldingapproximately 15% binding of the radiolabled probe peptide were used forall competitive inhibition assays. Under these conditions theconcentration of the labeled peptide is less than the concentration ofthe HLA molecule and the IC₅₀ is less than the concentration of the HLAmolecule, therefore the measured IC₅₀s are reasonable approximations ofthe true K_(D) values. Each competitor peptide was tested in two to fourcompletely independent experiments. As a positive control, in eachexperiment, the unlabeled version of the relevant radiolabeled probe wastested and its IC₅₀ measured. The average IC₅₀ Of A3CON1 for the A3,A11, A31, and A*6801 assays were 11, 6, 18, and 8 nM, respectively. Theaverage IC₅₀ of the HBVc 141-151 peptide in the A*3301 assay was 29 nM.

Example 2 Implementation of the Extended Supermotif to Improve theBinding Capacity of Native Peptides by Creating Analogs

HLA motifs and supermotifs (comprising primary and/or secondaryresidues) are useful in preparing highly cross-reactive native peptides,as demonstrated herein. Moreover, the definition of HLA motifs andsupermotifs also allows one to engineer highly cross-reactive epitopesby identifying residues within a native peptide sequence which can beanaloged, or “fixed”, to confer upon a peptide certain characteristics,e.g., greater cross-reactivity within the group of HLA molecules thatmake-up the supertype, and/or greater binding affinity for some or allof those HLA molecules Examples of analog peptides that exhibitmodulated binding affinity are provided.

Analogs representing primary anchor single amino acid residuessubstituted with I residues at the C-terminus of two different B7-likepeptides (HBV env 313 and HBV pol 541) were synthesized and tested fortheir B7-supertype binding capacity. It was found that the Isubstitution had an overall positive effect on binding affinity and/orcross-reactivity in both cases. In the case of HBV env 313 the 19 (I atC-terminal position 9) replacement was effective in increasingcross-reactivity from 4 to 5 alleles bound by virtue of an almost400-fold increase B*5401 binding affinity. In the case of HBV pol 541,increased cross-reactivity was similarly achieved by a substantialincrease in B*5401 binding. Also, significant gains in binding affinityfor B*0702, B51, and B*5301 were observed with the HBV pol 541 I9analog.

Moreover, HLA supermotifs are of value in engineering highlycross-reactive peptides by identifying particular residues at secondaryanchor positions that are associated with such cross-reactiveproperties. Demonstrating this, the capacity of a second set of peptidesrepresenting discreet single amino acid substitutions at positions oneand three of five different B7-supertype binding peptides weresynthesized and tested for their B-7 supertype binding capacity. In 4/4cases the effect of replacing the native residue at position 1 with thearomatic residue F (an “F1” substitution) resulted in an increase incross-reactivity, compared to the parent peptide, and, in mostinstances, binding affinity was increased three-fold or better (TableXXII). More specifically, for HBV env 313, MAGE2 170, and HCV core 168complete supertype cross-reactivity was achieved with the F1substitution analogs. These gains were achieved by dramaticallyincreasing B*5401 binding affinity. Also, gains in affinity were notedfor other alleles in the cases of HCV core 168((B*3501 and B*5301) andMAGE2 170((B*3501, B51 and B*5301). Finally, in the case of MAGE3 196,the F1 replacement was effective in increasing cross-reactivity becauseof gains in B*0702 binding. An almost 70-fold increase in B51 bindingcapacity was also noted.

Two analogs were also made using the supermotif positive F substitutionat position three (an “F3” substitution). In both instances increases inbinding affinity and cross-reactivity were achieved. Specifically, inthe case of HBV pol 541, the F3 substitution was effective in increasingcross-reactivity by virtue of its effect on B*5401 binding. In the caseof MAGE3 196, complete supertype cross-reactivity was achieved byincreasing B*0702 and B*3501 binding capacity. Also, in the case ofMAGE3 196, it is notable that increases in binding capacity between 40-and 5000-fold were obtained for B*3501, B51, B*5301, and B*5401.

In conclusion, these data demonstrate that by the use of even singleamino acid substitutions, it is possible to increase the bindingaffinity and/or cross-reactivity of peptide ligands for HLA supertypemolecules.

Example 3 Induction Of HLA-Restricted CTL By Subcutaneous Priming WithHBV Peptide In Incomplete Freund's Adjuvant (IFA)

The immunogenicity of HLA class I binding peptides can be assessed invivo as described in, e.g., Sette et al. J. Immunol. 153:5586-5592(1994). This example illustrates such a procedure, whereby subcutaneousinjection of HBV peptide in Incomplete Freund's Adjuvant (IFA) can beused to induce HBV-specific CTL in mice that are transgenic for a humanHLA allele such as the human HLA-A11 allele.

Priming and In Vitro Restimulation: Mice that are transgenic forHLA-A11, (e.g. HLA-A11/Kb strain) are injected with 100 microliters ofan emulsion of purified HBV peptide in IFA. The purified peptidecomprises an A11 motif, and is selected from the preferred peptideslisted in Table XVI or, alternatively, may be an analog peptide. Thepeptide epitope (50 μg/mouse) and equimolar amounts of the helperepitope HBV core 128-140 (140 μg/mouse) are dissolved in PBS/5% DMSO,emulsified in IFA, and injected subcutaneously at the base of the tailof the transgenic mice. Eleven days after priming, splenocytes (5×10⁶cells/well in a 24-well plate) obtained from these animals arerestimulated with syngeneic irradiated LPS blasts (2×10⁶/well) coatedwith peptide.

LPS blasts from unprimed HLA-A11 transgenic mice are prepared 72 hoursbefore use by suspending splenocytes in medium containing LPS (25 μg/ml)and dextran sulfate (7 μg/ml). Coating is achieved by incubating 50 μgof peptide with 1.2×10⁶ LPS blasts in a volume of 0.4 ml of RPMI mediumsupplemented with 10% FCS for 1 hour at 37° C. The cells are washed onceand then co-cultured with splenocytes. After six days, effector cellsare assayed, as outlined for example in Example 5, for cytotoxicityagainst ⁵¹Cr-labeled 3A4-721.221-A11K^(b) target cells in the presenceof the peptide.

The effector cells (2×10⁶ cells/well) are re-stimulated at weeklyintervals. For the first re-stimulation, peptide-coated LPS blasts areused, followed by peptide-coated A11/K^(b) cells. Six days afaterre-stimulation, effector cells are assayed for cytotoxicity as above.

Example 4 Recognition of Generation of Endogenous Processed AntigensAfter Priming

This example determines that CTL induced by in vivo priming with peptide(as disclosed in Example 3) recognize endogenously synthesized antigens.

Effector cells from the procedure disclosed in Example 3 arere-stimulated in vitro using peptide-coated stimulator cells. Six dayslater, effector cells are assayed for cytotoxicity and the cell linesthat contain peptide-specific cytotoxic activity are furtherre-stimulated. An additional six days later, these cell lines are testedfor cytotoxic activity on ⁵¹Cr labeled 3A4-721.221-A11/K^(b) targetcells, in the absence or presence of peptide, and also tested on ⁵¹Crlabeled target cells bearing the endogenously synthesized antigen.

The result will demonstrate that CTL lines obtained from animals primedwith peptide epitope recognize endogenously synthesized HBV antigen.

Example 5 Activity of CTL-HTL Conjugated Epitopes in Transgenic Mice

This example illustrates the induction of CTLs in transgenic mice by useof an HBV CTL/HTL peptide conjugate. An analagous study may be found inOseroff et al. Vaccine 16:823-833 (1998). The peptide composition cancomprise multiple CTL and/o HTL epitopes. Such a peptide composition cancomprise a lipidated HTL epitope conjugated to a preferred CTL epitopecontaining, for example, an A11 motif or an analog of that epitope.

Lipopeptides are prepared by coupling the appropriate fatty acid to theamino terminus of the resin bound peptide. A typical procedure is asfollows: A dichloromethane solution of a four-fold excess of apre-formed symmetrical anhydride of the appropriate fatty acid is addedto the resin and the mixture is allowed to react for two hours. Theresin is washed with dichloromethane and dried. The resin is thentreated with trifluoroacetic acid in the presence of appropriatescavengers [e.g. 5% (v/v) water] for 60 minutes at 20° C. Afterevaporation of excess trifluoroacetic acid, the crude peptide is washedwith diethyl ether, dissolved in methanol and precipitated by theaddition of water. The peptide is collected by filtration and dried.

Preparation of peptides for immunization: Peptide compositions aretypically resuspended in DMSO at a concentration of 20 mg/ml. Beforeuse, peptides are prepared at the required concentration by dilution insaline or the appropriate medium.

Immunization procedures: A11/Kb mice, which are transgenic for the humanHLA A11 allele, are primed subcutaneously (base of the tail) with 0.1 mlof peptide conjugate formulated in saline, or DMSO/saline. Seven daysafter priming, splenocytes obtained from these animals are restimulatedwith syngeneic irradiated LPS-activated lymphoblasts coated withpeptide.

Media:

a. RPMI-1640 supplemented with 10% fetal calf serum (FCS) 2 mMGlutamine, 50 μg/ml Gentamicin and 5×10⁻⁵ M 2-mercaptoethanol serves asculture medium

b. RPMI-1640 containing 25 mM HEPES buffer and supplemented with 2%(FCS) is used as cell washing medium.

Cell lines: The 3A4-721.221-A11/K^(b) cell line is used as target cells.This cell line is an EBV transformed cell line that was mutagenized andselected to be Class I negative which was transfected with anHLA-A11/K^(b) gene.

LPS-activated lymphoblasts: Splenocytes obtained from transgenic miceare resuspended at a concentration of 1-1.5×10⁶/ml in culture mediumsupplemented with 25 μg/ml LPS and 7 μg/ml dextran sulfate in 75 cmtissue culture flasks. After 72 hours at 37° C., the lymphoblasts arecollected for use by centrifugation.

Peptide coating of lymphoblasts: Peptide coating of the LPS activatedlymphoblasts is achieved by incubating 30×10⁶ irradiated (3000 rads)lymphoblasts with 100 μg of peptide in 1 ml of R10 medium for 1 hr at37° C. Cells are then washed once and resuspended in culture medium atthe desired concentration.

In vitro CTL activation: One week after priming, spleen cells (30×10⁶cells/flask) are co-cultured at 37° C. with syngeneic, irradiated (3000rads), peptide coated lymphoblasts (10×10⁶ cells/flask) in 10 ml ofculture medium/T25 flask. After six days, the effector cells areharvested and assayed for cytotoxic activity.

Assay for cytotoxic activity: Target cells (1.0-1.5×10⁶) are incubatedat 37° C. in the presence of 200 μl of sodium ⁵¹Cr chromate. After 60minutes, cells are washed three times and resuspended in R10 medium.Peptide is added where required at a concentration of 1 μg/ml. For theassay, 104 ⁵¹Cr-labeled target cells are added to differentconcentrations of effector cells (final volume of 200 μl) in U-bottom96-well plates. After a 6 hour incubation period at 37° C., a 0.1 mlaliquot of supernatant is removed from each well and radioactivity isdetermined in a Micromedic automatic gamma counter. The percent specificlysis is determined by the formula: percent specificrelease=100×(experimental release−spontaneous release)/(maximumrelease−spontaneous release). To facilitate comparison between separateCTL assays run under the same conditions, % ⁵¹Cr release data isexpressed as lytic units/10⁶ cells. One lytic unit is arbitrarilydefined as the number of effector cells required to achieve 30% lysis of10,000 target cells in a 6 hour SiCr release assay. To obtain specificlytic units/10⁶, the lytic units/10⁶ obtained in the absence of peptideis subtracted from the lytic units/10⁶ obtained in the presence ofpeptide. For example, if 30% ⁵¹Cr release is obtained at the E:T of 50:1(i.e., 5×10⁵ effector cells for 10,000 targets) in the absence ofpeptide and 5:1 (i.e., 5×10⁴ effector cells for 10,000 targets) in thepresence of peptide, the specific lytic units would be:(1×10⁶(5×10⁴)−(1×10⁶(5×10⁵)=18 LU/10⁶.

The results are analyzed to assess the magnitude of the CTL responses ofanimals injected with the immunogenic CTL/HTL conjugate vaccinepreparation. Analyses similar to this may be performed to evaluate theimmunogenicity of peptide conjugates containing multiple CTL epitopesand/or multiple HTL epitopes. In accordance with these procedures it isfound that CTL and HTL responses are induced.

Example 7 Induction Of Specific CTL Response In Humans

A human clinical trial for an immunogenic composition comprising CTL andHTL epitopes is set up as an IND Phase I, dose escalation study (5, 50and 500 μg) and carried out as a randomized, double-blind,placebo-controlled trial. Such a trial is designed, for example, asfollows:

A total of about 27 subjects are enrolled and divided into 3 groups:

-   -   Group I: 3 subjects are injected with placebo and 6 subjects are        injected with 5 μg of peptide composition;    -   Group II: 3 subjects are injected with placebo and 6 subjects        are injected with 50 μg peptide composition;    -   Group III: 3 subjects are injected with placebo and 6 subjects        are injected with 500 μg of peptide composition.

After 4 weeks following the first injection, all subjects receive abooster inoculation at the same dosage.

The endpoints measured in this study relate to the safety andtolerability of the peptide composition as well as its immunogenicity.Cellular immune responses to the peptide composition are an index of theintrinsic activity of this the peptide composition, and can therefore beviewed as a measure of biological efficacy. The following summarize theclinical and laboratory data that relate to safety and efficacyendpoints.

Safety: The incidence of adverse events is monitored in the placebo anddrug treatment group and assessed in terms of degree and reversibility.

Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy,subjects are bled before and after injection. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

Thus, the vaccine is found to be both safe and efficacious.

Example 8 Phase II Trials in Patients Infected with HBV

Phase II trials are performed to study the effect of administering theCTL-HTL peptide compositions to patients (male and female) havingchronic HBV infection. A main objective of the trials is to determine aneffective dose and regimen for inducing CTLs in chronically infected HBVpatients, to establish the safety of inducing a CTL response in thesepatients, and to see to what extent activation of CTLs improves theclinical picture of chronically infected CTL patients, as manifested bya transient flare in alanine aminotransferase (ALT), normalization ofALT, and reduction in HBV DNA. Such a study is designed, for example, asfollows:

The studies are performed in multiple centers in the U.S. and Canada.The trial design is an open-label, uncontrolled, dose escalationprotocol wherein the peptide composition is administered as a singledose followed six weeks later by a single booster shot of the same dose.The dosages are 50, 500 and 5,000 micrograms per injection.Drug-associated adverse effects are recorded.

There are three patient groupings. The first group is injected with 50micrograms of the peptide composition and the second and third groupswith 500 and 5,000 micrograms of peptide composition, respectively. Thepatients within each group range in age from 21-65 and include bothmales and females. The patients represent diverse ethnic backgrounds.All of them are infected with HBV for over five years and are HIV, HCVand HDV negative, but have positive levels of HBe antigen and HBsantigen.

The magnitude and incidence of ALT flares and the levels of HBV DNA inthe blood are monitored to assess the effects of administering thepeptide compositions. The levels of HBV DNA in the blood are an indirectindication of the progress of treatment. The vaccine composition isfound to be both safe and efficacious in the treatment of chronic HBVinfection.

Example 9 Selection of CTL and HTL Epitopes for Inclusion in anHBV-Specific Vaccine

This example illustrates the procedure for the selection of peptideepitopes for vaccine compositions of the invention.

The following principles are utilized when selecting an array ofepitopes for inclusion in a polyepitopic composition, or for selectingepitopes to be included in a vaccine composition and/or to be encoded bya minigene. Each of the following principles are balanced in order tomake the selection.

1.) Epitopes are selected which, upon administration, mimic immuneresponses that have been observed to be correlated with HBV clearance.For HLA Class I this includes 3-4 epitopes that come from at least oneantigen of HBV. In other words, it has been observed that in patientswho spontaneously clear HBV, that they had generated an immune responseto at least 3 epitopes on at least one HBV antigen. For HLA Class II asimilar rationale is employed; again 3-4 epitopes are selected from atleast one HBV antigen.

2.) Epitopes are selected that have the requisite binding affinityestablished to be correlated with immunogenicity: for HLA Class I anIC₅₀ of 500 nM or less, or for Class II an IC₅₀ of 1000 nM or less.

3.) Sufficient supermotif bearing peptides, or a sufficient array ofallele-specific motif bearing peptides, are selected to give broadpopulation coverage. For example, epitopes are selected to provide atleast 80% population coverage. A Monte Carlo analysis, a statisticalevaluation known in the art, is employed to assess population coverage.

4.) When selecting epitopes for HBV antigens it is often preferable toselect native epitopes. Therefore, of particular relevance forinfectious disease vaccines, are epitopes referred to as “nestedepitopes.” Nested epitopes occur where at least two epitopes overlap ina given peptide sequence. A peptide comprising “transcendent nestedepitopes” is a peptide that has both HLA class I and HLA class IIepitopes in it.

When providing nested epitopes, a sequence that has the greatest numberof epitopes per provided sequence is provided. A limitation on thisprinciple is to avoid providing a peptide that is any longer than theamino terminus of the amino terminal epitope and the carboxyl terminusof the carboxyl terminal epitope in the peptide. When providing a longerpeptide sequence, such as a sequence comprising nested epitopes, thesequence is screened in order to insure that it does not havepathological or other deleterious biological properties.

5.) When creating a minigene, as disclosed in greater detail in theExample 10, an objective is to generate the smallest peptide possiblethat encompasses the epitopes of interest. The principles employed aresimilar, if not the same as those employed when selecting a peptidecomprising nested epitopes. Thus, upon determination of the nucleic acidsequence to be provided as a minigene, the peptide encoded thereby isanalyzed to determine whether any “junctional epitopes” have beencreated. A junctional epitope is an actual binding epitope, aspredicted, e.g., by motif analysis. Junctional epitopes are to beavoided because the recipient may generate an immune response to thatepitope. Of particular concern is a junctional epitope that is a“dominant epitope.” A dominant epitope may lead to such a zealousresponse that immune responses to other epitopes are diminished orsuppressed.

Peptide epitopes for inclusion in vaccine compositions are, for example,selected from those lsited in Table XXIII. A vaccine compositioncomprised of selected peptides, when administered, is safe, efficacious,and elicits an immune response similar in magnitude of an immuneresponse that clears an acute HBV infection.

Example 10 Construction of Minigene Multi-Epitope DNA Pslasmids

Expression plasmids have been constructed and evaluated as described,for example, in U.S. Ser. No. 60/085,751 filed May 15, 1998 and U.S.Ser. No. 09/078,904 filed May 13, 1998. The binding peptide epitopes andtheir positions for some of the plasmids described therein are shown inFIG. 1 as example of the orientation of peptide epitopes in minigeneconstructs. Such a plasmid may, for example, also include multiple CTLand HTL peptide epitopes. In the present example, HLA-A11 motif-bearingpeptides are used in conjunction with DR supermotif-bearing peptides.Preferred A11 epitopes are identified, for example, in Table XVI orTable XXI and peptide epitopes recognized by HLA DR molecules (TablesXVIII and XIX). Four class I A11 motif-bearing peptide epitopes oranalogs of those peptide epitopes derived from the same HBV antigen,e.g. the envelope protein, are selected as CTL epitopes. Four class IImotif-bearing peptide epitopes derived from the same antigen, e.g., theenvelope protein, are selected as HTL epitopes. These epitopes are thenincorporated into a minigene for expression in an expression vector.

This example illustrates the methods to be used for construction of sucha minigene-bearing expression plasmid. Other expression vectors that maybe used for minigene compositions are available and known to those ofskill in the art.

A pMin minigene DNA plasmid is constructed from an early generation DNAplasmid designated as pMin.0. This plasmid contains a consensus Kozaksequence and a consensus murine kappa Ig-light chain signal sequencefollowed by a string of CTL and HTL epitopes selected in accordance withprinciples disclosed herein. The pMIN sequence encodes an open readingframe fused to the Myc and His antibody epitope tag coded for by thepcDNA 3.1 Myc-His vector.

Overlapping oligonucleotides, for example eight oligonucleotides,averaging approximately 70 nucleotides in length with 15 nucleotideoverlaps, are synthesized and HPLC-purified. The oligonucleotides encodethe selected peptide epitopes as well as appropriate linker nucleotides.The final multiepitope minigene is assembled by extending theoverlapping oligonucleotides in three sets of reactions using PCR. APerkin/Elmer 9600 PCR machine is used and a total of 30 cycles areperformed using the following conditions: 95° C. for 15 sec, annealingtemperature (5° below the lowest calculated Tm of each primer pair) for30 sec, and 72° C. for 1 min.

For the first PCR reaction, 5 μg of each of two oligonucleotides areannealed and extended: Oligonucleotides 1+2, 3+4, 5+6, and 7+8 arecombined in 100 μl reactions containing Pfu polymerase buffer (1×=10 mMKCL, 10 mM (NH₄)₂SO₄, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO₄, 0.1%Triton X-100, 100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfupolymerase. The full-length dimer products are gel-purified, and tworeactions containing the product of 1+2 and 3+4, and the product of 5+6and 7+8 are mixed, annealed, and extended for 10 cycles. Half of the tworeactions are then mixed, and 5 cycles of annealing and extensioncarried out before flanking primers are added to amplify the full lengthproduct for 25 additional cycles. The full-length product isgel-purified and cloned into pCR-blunt (Invitrogen) and individualclones are screened by sequencing.

Example 11 The plasmid construct and the degree to which it inducesimmunogenicity

The degree to which the plasmid construct prepared using the methodologyoutlined in Example 10 is able to induce immunogenicity is evaluatedthrough in vivo injections into mice and in vitro CTL culture andcytotoxicity assays as detailed e.g., in U.S. Ser. No. 60/085,751 filedMay 15, 1998. To assess the capacity of the pMin minigene construct toinduce CTLs in vivo, HLA-A11/K^(b) transgenic mice are immunizedintramuscularly with 100 μg of naked cDNA. As a means of comparing thelevel of CTLs induced by cDNA immunization, a control group of animalsis also immunized with an actual peptide composition that comprisesmultiple epitopes synthesized as a single polypeptide.

Splenocytes from immunized animals are stimulated twice with each of thepeptide epitopes encoded in the minigene, then assayed forpeptide-specific cytotoxic activity in a ⁵¹Cr release assay. The resultsindicate the magnitude of the CTL response directed against each of itsA11-restricted epitopes, thus indicating the in vivo immunogenicity ofthe minigene vaccine. It is, therefore, found that the minigene elicitsimmune responses directed toward A11-restricted epitopes.

Example 12 Peptide Composition for Prophylactic Uses

Vaccine compositions of the present invention are used to prevent HBVinfection in persons who are at risk. For example, a polyepitopicpeptide epitope composition containing multiple CTL and HTL epitopessuch as those selected in Examples 9 and/or 10, which are also selectedto target greater than 80% of the population, is administered toindividuals at risk for HBV infection. The composition is provided as asingle lipidated polypeptide that encompasses multiple epitopes. Thevaccine is administered in an aqueous carrier comprised of FreundsIncomplete Adjuvant. The dose of peptide for the initial immunization isfrom about 1 to about 5,000 μg for a 70 kg patient. The initialadministration of vaccine is followed by booster dosages at 4 weeksfollowed by evaluation of the magnitude of the immune response in thepatient by techniques that determine the presence of epitope-specificCTL populations in a PBMC sample. Additional booster doses areadministered as required. The composition is found to be both safe andefficacious as a prophylaxis against HBV infection.

Alternatively, the polyepitopic peptide composition can be administeredas a nucleic acid in accordance with methodologies known in the art anddisclosed herein.

Example 13 Polyepitopic Vaccine Compositions Derived from Native HBVSequences

A native HBV polyprotein sequence is screened, preferably using computeralgorithms defined for each class I and/or class II supermotif or motif,to identify “relatively short” regions of the polyprotein that comprisemultiple epitopes. This relatively short sequence that contains multipledistinct, even overlapping, epitopes is selected and used to generate aminigene construct. The construct is engineered to express the peptide,which corresponds to the native protein sequence. The “relatively short”peptide is less than 100 amino acids in length, preferably less than 75amino acids in length, and more preferably less than 50 amino acids inlength. The protein sequence of the vaccine composition is selectedbecause it has maximal number of epitopes contained within the sequence.As noted herein, epitope motifs may be overlapping (i.e., frame shiftedrelative to one another) with frame shifted overlapping epitopes, e.g.two 9-mer epitopes can be present in a 10 amino acid peptide. Such avaccine composition is administered for therapeutic or prophylacticpurposes.

The vaccine composition will preferably include, for example, three CTLepitopes and at least one HTL epitope from the source antigen.Junctional sequences will be analyzed to avoid sequences containing apotentially immunodominant epitope. This polyepitopic native sequence isadministered either as a peptide or as a nucleic acid sequence whichencodes the peptide. Alternatively, an analog can be made of this nativesequence.

The embodiment of this example provides for the possibility that an asyet undiscovered aspect of immune system processing will apply to thenative nested sequence and thereby facilitate the production oftherapeutic or prophylactic immune response-inducing vaccinecompositions. Additionally such an embodiment provides for thepossibility of motif-bearing epitopes for an HLA makeup that ispresently unknown. Furthermore, this embodiment directs the immuneresponse to sequences that are present in native HBV antigens. Lastly,the embodiment provides an economy of scale when producing nucleic acidvaccine compositions.

Related to this embodiment, computer programs can be derived whichidentify, in a target sequence, the greatest number of epitopes persequence length.

Example 14 Polyepitotpic Vaccine Compositions Directed to MultipleDiseases

The HIBV peptide epitopes of the present invention are used inconjunction with peptide epitopes from target antigens related to one ormore other diseases, to create a vaccine composition that is useful forthe prevention or treatment of HBV as well as another disease. Examplesof other diseases include, but are not limited to, HIV, HCV, and HPV.

For example, a polyepitopic peptide composition comprising multiple CTLand HTL epitopes that target greater than 98% of the population may becreated for administration to individuals at risk for both HBV and HIVinfection. The composition can be provided as a single polypeptide thatincorporates the multiple epitopes from the various disease-associatedsources.

Example 15 Use of Peptides to Evaluate an Immune Response

Peptides of the invention may be used to analyze an immune response forthe presence of specific CTL populations corresponding to HBV. Such ananalysis may be performed as described by Ogg et al., Science279:2103-2106, 1998. In the following example, peptides in accordancewith the invention are used as a reagent for diagnostic or prognosticpurposes, not as an immunogen.

In this example highly sensitive human leukocyte antigen tetramericcomplexes (“tetramers”) may be used for a cross-sectional analysis of,for example, HBV Env-specific CTL frequencies from untreated HLAA*0201-positive indiviuals at different stages of infection using an HBVEnv peptide containing an A2.1 extended motif. Tetrameric complexes aresynethesized as described (Musey et al., N. Engl. J. Med. 337:1267,1997). Briefly, purified HLA heavy chain (A2.1 in this example) andβ2-microglobulin are synthesized by means of a prokaryotic expressionsystem. The heavy chain is modified by deletion of thetransmembrane-cytosolic tail and COOH-terminal addition of a sequencecontaining a BirA enzymatic biotinylation site. The heavy chain,β2-microglobulin, and peptide are refolded by dilution. The 45-kDrefolded product is isolated by fast protein liquid chromatography andthen biotinylated by BirA in the presence of biotin (Sigma, St. Louis,Mo.), adenosine 5′triphosphate and magnesium. Streptavidin-phycoerythrinconjugate is added in a 1:4 molar ratio, and the tetrameric product isconcentrated to 1 mg/ml. The resulting product is referred to astetramer-phycoerythrin.

Approximately one million PBMCs are centrifuged at 300 g for 5 minutesand resuspended in 50 ul of cold phosphate-buffered saline. Tri-coloranalysis is performed with the tetramer-phycoerythrin, along withanti-CD8-Tricolor, and anti-CD38. The PBMCs are incubated with tetramerand antibodies on ice for 30 to 60 min and then washed twice beforeformaldehyde fixaation. Gates are applied to contain >99.98% of controlsamples. Controls for the tetramers include both A*0201-negativeindividuals and A*0201-positive uninfected donors. The percentage ofcells stained with the tetramer is then determined by flow cytometry.The results indicate the number of cells in the PBMC sample that containepitope-restricted CTLs, thereby readily indicating the stage ofinfection with HBV or the status of exposure to HBV or to a vaccine thatelicits a protective response.

Example 16 Use of Peptide Epitopes to Evaluate Recall Responses

The peptide epitopes of the invention are used as reagents to evaluate Tcell responses such as acute or recall responses, in patients. Such ananalysis may be performed on patients who have recovered from infectionor who are chronically infected with HBV or who have been vaccinatedwith an HBV vaccine.

For example, the class I restricted CTL response of persons at risk forHBV infection who have been vaccinated may be analyzed. The vaccine maybe any HBV vaccine. PBMC are collected from vaccinated individuals andHLA typed. Appropriate peptide reagents that, are highly conserved and,optimally, bear supermotifs to provide cross-reactivity with multipleHLA supertype family members are then used for analysis of samplesderived from individuals who bear that HLA type.

PBMC from vaccinated individuals are separated on Ficoll-Histopaquedensity gradients (Sigma Chemical Co., St. Louis, Mo.), washed threetimes in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCOLaboratories) supplemented with L-glutamine (2 mM), penicillin (50U/ml), streptomycin (50 μg/ml), and Hepes (10 mM) containing 10%heat-inactivated human AB serum (complete RPMI) and plated usingmicroculture formats. Synthetic peptide is added at 10 μg/ml to eachwell and recombinant HBc Ag is added at 1 μg/ml to each well as a sourceof T cell help during the first week of stimulation.

In the microculture format, 4×10⁵ PBMC are stimulated with peptide in 8replicate cultures in 96-well round bottom plate in 100 μl/well ofcomplete RPMI. On days 3 and 10, 100 ml of complete RPMI and 20 U/mlfinal concentration of rIL-2 are added to each well. On day 7 thecultures are transferred into a 96-well flat-bottom plate andrestimualted with peptide, rIL-2 and 10⁵ irradiated (3,000 rad)autologous feeder cells. The cultures are tested for cytotoxic activityon day 14. A positive CTL response requires two or more of the eightreplicate cultures to display greater than 10% specific ⁵¹Cr release,based on comparison with uninfected control subjects as previouslydescribed (Rehermann, et al., Nature Med. 2:1104,1108, 1996; Rehermannet al., J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al. J.Clin. Invest. 98:1432-1440, 1996).

Target cell lines are autologous and allogeneic EBV-transformed B-LCLthat are either purchased from the American Society forHistocompatibility and Immunogenetics (ASHI, Boston, Mass.) orestablished from the pool of patients as described (Guilhot, et al. J.Virol. 66:2670-2678, 1992).

Cytotoxicity assays are performed in the following manner. Target cellsconsist of either allogeneic HLA-matched or autologous EBV-transformed Blymphoblastoid cell line that are incubated overnight with syntheticpeptide at 10 μM and labeled with 100 μCi of ⁵¹Cr (Amersham Corp.,Arlington Heights, Ill.) for 1 hour after which they are washed fourtimes with HBSS. Cytolytic activity is determined in a standard 4-h,split well ⁵¹Cr release assay using U-bottomed 96 well plates containing3,000 targets/well. Stimulated PBMC are tested at E/T ratios of 20-50:1on day 14. Percent cytotoxicity is determined from the formula:100×[(experimental release-spontaneous release)/maximumrelease-spontaneous release)]. Maximum release is determined by lysis oftargets by detergent (2% Triton X-100; Sigma Chemical Co., St. Louis,Mo.). Spontaneous release is <25% of maximum release for allexperiments.

The results of such an analysis will indicate to what extentHLA-restricted CTL populations have been stimulated with the vaccine. Ofcourse, this protocol can also be used to monitor prior HBV exposure.

The above examples are provided to illustrate the invention but not tolimit its scope. For example, the human terminology for the MajorHistocompatibility Complex, namely HLA, is used throughout thisdocument. It is to be appreciated that these principles can be extendedto other species as well. Moreover, peptide epitopes have been disclosedin the related application U.S. Ser. No. 08/820,360, which waspreviously incorporated by reference. Thus, other variants of theinvention will be readily apparent to one of ordinary skill in the artand are encompassed by the appended claims. All publications, patents,and patent application cited herein are hereby incorporated by referencefor all purposes. TABLE II POSITION SUPER- MOTIFS 1 2 3 4 5 6 7 8C-terminus A1 $\frac{1{^\circ}\quad{Anchor}}{{TI}{LVMS}}$$\frac{1{^\circ}\quad{Anchor}}{FWY}$ A2$\frac{1{^\circ}\quad{Anchor}}{{LIVM}{ATQ}}$$\frac{1{^\circ}\quad{Anchor}}{LIVMAT}$ A3 preferred$\frac{1{^\circ}\quad{Anchor}}{{VSMA}{TL}I}$ YFW (4/5) YFW (3/5) YFW(4/5) P (4/5) $\frac{1{^\circ}\quad{Anchor}}{RK}$ deleterious DE (3/5);DE (4/5) P (5/5) A24 $\frac{1{^\circ}\quad{Anchor}}{{YF}{WIVLMT}}$$\frac{1{^\circ}\quad{Anchor}}{{FI}{YWLM}}$ B7 preferred FWY (5/5) LIVM(3/5) $\frac{1{^\circ}\quad{Anchor}}{P}$ FWY (4/5) FWY (3/5)$\frac{1{^\circ}\quad{Anchor}}{{VILF}{MWYA}}$ deleterious DE (3/5); DE(3/5) G (4/5) QN (4/5) DE (4/5) P (5/5); G (4/5); A (3/5); QN (3/5) B27$\frac{1{^\circ}\quad{Anchor}}{RHK}$$\frac{1{^\circ}\quad{Anchor}}{{FYL}{WMI}}$ B44$\frac{1{^\circ}\quad{Anchor}}{ED}$$\frac{1{^\circ}\quad{Anchor}}{FWYLIMVA}$ B58$\frac{1{^\circ}\quad{Anchor}}{ATS}$$\frac{1{^\circ}\quad{Anchor}}{{FWY}{LIV}}$ B62$\frac{1{^\circ}\quad{Anchor}}{{QL}{IVMP}}$$\frac{1{^\circ}\quad{Anchor}}{{FWY}{MIV}}$ A1 9-mer preferred GFYW$\frac{1{^\circ}\quad{Anchor}}{STM}$ DEA YFW P DEQN YFW$\frac{1{^\circ}\quad{Anchor}}{Y}$ deleterious DE RHKLIVM A H A P A19-mer preferred GRHK ASTCLIV M $\frac{1{^\circ}\quad{Anchor}}{{DE}{AS}}$GSTC ASTC LIVM DE $\frac{1{^\circ}\quad{Anchor}}{Y}$ deleterious ARHKDEPY DE PQN RHK PG GP FW POSITION 9 or POSITION: 1 2 3 4 5 6 7 8C-terminus C-terminus A1 9-mer preferred YFW$\frac{1{^\circ}\quad{Anchor}}{STM}$ DEAQN A YFWQN PASTC GDE P$\frac{1{^\circ}\quad{Anchor}}{Y}$ deleterious GP RHKGLIV DE RHK QNARHKYFW RHK A M A1 10-mer preferred YFW STCLIVM$\frac{1{^\circ}\quad{Anchor}}{{DE}{AS}}$ A YFW PG G YFW$\frac{1{^\circ}\quad{Anchor}}{Y}$ deleterious RHK RHKDEPY P G PRHK QNFW A2.1 9-mer preferred YFW $\frac{1{^\circ}\quad{Anchor}}{{LM}{IVQAT}}$YFW STC YFW A P $\frac{1{^\circ}\quad{Anchor}}{V{LIMAT}}$ deleteriousDEP DERKH RKH DERKH A2.1 10-mer preferred AYFW$\frac{1{^\circ}\quad{Anchor}}{{LM}{IVQAT}}$ LVIM G G FYWL VIM$\frac{1{^\circ}\quad{Anchor}}{V{LIMAT}}$ deleterious DEP DE RKHA P RKHDERKLH RKH A3 preferred RHK$\frac{1{^\circ}\quad{Anchor}}{{LMVISATF}{CGD}}$ YFW PRHKK YFW A YFW P$\frac{1{^\circ}\quad{Anchor}}{{KYR}{HFA}}$ deleterious DEP DE A11preferred A $\frac{1{^\circ}\quad{Anchor}}{{VTLMISAGN}{CDF}}$ YFW YFW AYFW YFW P $\frac{1{^\circ}\quad{Anchor}}{K{RYH}}$ deleterious DEP A GA24 9-mer preferred YFWRHK $\frac{1{^\circ}\quad{Anchor}}{{YFW}M}$ STCYFW YFW $\frac{1{^\circ}\quad{Anchor}}{FLIW}$ deleterious DEG DE G QNPDERH G AQN K A24 10-mer preferred$\frac{1{^\circ}\quad{Anchor}}{{YFW}M}$ P YFWP P$\frac{1{^\circ}\quad{Anchor}}{FLIW}$ deleterious GDE QN RHK DE A QN DEAA3101 preferred RHK $\frac{1{^\circ}\quad{Anchor}}{{MVT}{ALIS}}$ YFW PYFW YFW AP $\frac{1{^\circ}\quad{Anchor}}{RK}$ deleterious DEP DE ADE DEDE DE A3301 preferred $\frac{1{^\circ}\quad{Anchor}}{{MVALF}{IST}}$ YFWAYFW $\frac{1{^\circ}\quad{Anchor}}{RK}$ deleterious GP DE A6801preferred YFWSTC $\frac{1{^\circ}\quad{Anchor}}{{AVT}{MSLI}}$ YFWLIV MYFW P $\frac{1{^\circ}\quad{Anchor}}{RK}$ deleterious GP DEG RHK A B0702preferred RHKFW Y $\frac{1{^\circ}\quad{Anchor}}{P}$ RHK RHK RHK RHK PA$\frac{1{^\circ}\quad{Anchor}}{{LMF}{WYAIV}}$ deleterious DEQNP DEP DEDE GDE QN DE B3501 preferred FWYLIV M $\frac{1{^\circ}\quad{Anchor}}{P}$FWY $\frac{1{^\circ}\quad{Anchor}}{{LMFWY}{IVA}}$ deleterious AGP G GB51 preferred LIVMFW Y $\frac{1{^\circ}\quad{Anchor}}{P}$ FWY STC FWY GFWY $\frac{1{^\circ}\quad{Anchor}}{{LIVF}{WYAM}}$ deleterious AGPDERHKSTC DE G DEQN GDE B5301 preferred LIVMFW Y$\frac{1{^\circ}\quad{Anchor}}{P}$ FWY STC FWY LIVMFWY FWY$\frac{1{^\circ}\quad{Anchor}}{{IMFWY}{ALV}}$ deleterious AGPQN G RHKQNDE B5401 preferred FWY $\frac{1{^\circ}\quad{Anchor}}{P}$ FWYL IVM LIVMALIVM FWYAP $\frac{1{^\circ}\quad{Anchor}}{{ATIVL}{MFWY}}$ deleteriousGPQNDE GDES TC RHKDE DE QNDGE DEItalicized residues indicate less preferred or “tolerated” residues.The information in this Table II specific for 9-mers unless otherwisespecified.

Italicized residues indicate less preferred or “tolerated” residues.

The information in Table II is specific for 9-mers unless otherwisespecified. TABLE III Page 1 of 1 POSITION MOTIFS

DR4 preferred FMYLIVW M T I VSTCPALIM MH MH deleterious W R WDE DR1preferred MFLIVWY PAMQ VMATSPLIC M AVM deleterious C CH FD CWD GDE D DR7preferred MFLIVWY M W A IVMSACTPL M IV deleterious C G GRD N G DRSupermotif MFLIVWY VMSTACPLI DR3 MOTIFS

motif a LIVMFY D preferred motif b LIVMFAY DNQEST KRH preferredItalicized residues indicate less preferred or “tolerated” residues.

TABLE IV HLA Class I Standard Peptide Binding Affinity. STANDARDSTANDARD BINDING AFFINITY ALLELE PEPTIDE SEQUENCE (nM) A*0101 944.02YLEPAIAKY 25 A*0201 941.01 FLPSDYFPSV 5.0 A*0202 941.01 FLPSDYFPSV 4.3A*0203 941.01 FLPSDYFPSV 10 A*0206 941.01 FLPSDYFPSV 3.7 A*0207 941.01FLPSDYFPSV 23 A*6802 1141.02 FTQAGYPAL 40 A*0301 941.12 KVFPYALINK 11A*1101 940.06 AVDLYHFLK 6.0 A*3101 941.12 KVFPYALINK 18 A*3301 1083.02STLPETYVVRR 29 A*6801 941.12 KVFPYALINK 8.0 A*2401 979.02 AYIDNYNKF 12B*0702 1075.23 APRTLVYLL 5.5 B*3501 1021.05 FPFKYAAAF 7.2 B51 1021.05FPFKYAAAF 5.5 B*5301 1021.05 FPFKYAAAF 9.3 B*5401 1021.05 FPFKYAAAF 10

TABLE V HLA Class II Standard Peptide Binding Affinity. Binding Nomen-Standard Affinity Allele clature Peptide Sequence (nM) DRB1*0101 DR1515.01 PKYVKQNTLKLAT 5.0 DRB1*0301 DR3 829.02 YKTIAFDEEARR 300 DRB1*0401DR4w4 515.01 PKYVKQNTLKLAT 45 DRB1*0404 DR4w14 717.01 YARFQSQTTLKQKT 50DRB1*0405 DR4w15 717.01 YARFQSQTTLKQKT 38 DRB1*0701 DR7 553.01QYIKANSKFIGITE 25 DRB1*0802 DR8w2 553.01 QYIKANSKFIGITE 49 DRB1*0803DR8w3 553.01 QYIKANSKFIGITE 1600 DRB1*0901 DR9 553.01 QYIKANSKFIGITE 75DRB1*1101 DR5w11 553.01 QYIKANSKFIGITE 20 DRB1*1201 DR5w12 1200.05EALIHQLKINPYVLS 298 DRB1*1302 DR6w19 650.22 QYIKANAKFIGITE 3.5 DRB1*1501DR2w2β1 507.02 GRTQDENPVVHFFK 9.1 NIVTPRTPPP DRB3*0101 DR52a 511NGQIGNDPNRDIL 470 DRB4*0101 DRw53 717.01 YARFQSQTTLKQKT 58 DRB5*0101DR2w2β2 553.01 QYIKANSKFIGITE 20The “Nomenclature” column lists the allelic designations used in TableXVIII.

TABLE VI HBV A01 SUPER MOTIF (With binding information) ConservancyFreq. Protein Position Sequence String Peptide Filed A*0101 95 19 POL521 AICSVVRRAF XIXXXXXXXF 95 19 NUC 54 ALRQAILCW XLXXXXXXW 80 16 ENV 108AMQWNSTTF XMXXXXXXF 100 20 POL 166 ASFCGSPY XSXXXXXY 26.0026 100 20 POL166 ASFCGSPYSW XSXXXXXXXW 90 18 NUC 19 ASKLCLGW XSXXXXXW 85 17 NUC 19ASKLCLGWLW XSXXXXXXXW 80 16 POL 822 ASPLHVAW XSXXXXXW 100 20 ENV 312CIPIPSSW XIXXXXXW 100 20 ENV 312 CIPIPSSWAF XIXXXXXXXF 95 19 ENV 253CLIFLLVLLDY XLXXXXXXXXY 26.0548 95 19 ENV 239 CLRRFIIF XLXXXXXF 75 15ENV 239 CLRRFIIFLF XLXXXXXXXF 95 19 POL 523 CSVVRRAF XSXXXXXF 100 20 ENV310 CTCIPIPSSW XTXXXXXXXW 90 18 NUC 31 DIDPYKEF XIXXXXXF 85 17 NUC 29DLLDTASALY XLXXXXXXXY 1.0519 * 11.1000 95 19 ENV 196 DSWWTSLNF XSXXXXXXF20.0120 95 19 NUC 43 ELLSFLPSDF XLXXXXXXXF 95 19 NUC 43 ELLSFLPSDFFXLXXXXXXXXF 95 19 POL 374 ESRLVVDF XSXXXXXF 95 19 POL 374 ESRLVVDFSQFXSXXXXXXXXF 80 16 ENV 248 FILLLCLIF XIXXXXXXF 80 16 ENV 246 FLFILLLCLIFXLXXXXXXXXF 95 19 ENV 256 FLLVLLDY XLXXXXXY 26.0027 95 19 POL 658FSPTYKAF XSXXXXXF 90 18 X 63 FSSAGPCALRF XSXXXXXXXXF 100 20 ENV 333FSWLSLLVPF XSXXXXXXXF 20.0263 95 19 POL 656 FTFSPTYKAF XTXXXXXXXF20.0262 95 19 ENV 346 FVGLSPTVW XVXXXXXXW 95 19 POL 627 GLLGFAAPFXLXXXXXXF 20.0124 95 19 POL 509 GLSPFLLAQF XLXXXXXXXF 85 17 NUC 29GMDIDPYKEF XMXXXXXXXF 26.0372 95 19 NUC 123 GVWIRTPPAY XVXXXXXXXY 1.05250.0017 75 15 POL 569 HLNPNKTKRW XLXXXXXXXW 80 16 POL 491 HLYSHPIILGFXLXXXXXXXXF 85 17 POL 715 HTAELLAACF XTXXXXXXXF 95 19 NUC 52 HTALRQAILCWXTXXXXXXXXW 100 20 POL 149 HTLWKAGILY XTXXXXXXXY 1.0542 * 0.0300 100 20ENV 249 ILLLCLIF XLXXXXXF 80 16 POL 760 ILRGTSFVY XLXXXXXXY 1.0205 *0.0017 90 18 ENV 188 ILTIPQSLDSW XLXXXXXXXXW 90 18 POL 625 IVGLLGFAAPFXVXXXXXXXXF 80 16 POL 503 KIPMGVGLSPF XIXXXXXXXXF 85 17 NUC 21 KLCLGWLWXLXXXXXW 75 15 POL 108 KLIMPARF XLXXXXXF 75 15 POL 108 KLIMPARFYXLXXXXXXY 1.0171 0.0017 80 16 POL 610 KLPVNRPIDW XLXXXXXXXW 85 17 POL574 KTKRWGYSLNF XTXXXXXXXXF 95 19 POL 55 KVGNFTGLY XVXXXXXXY 1.0166 *0.0680 95 19 ENV 254 LIFLLVLLDY XIXXXXXXXY 1.0899 * 0.0084 100 20 POL109 LIMPARFY XIXXXXXY 26.0028 85 17 NUC 30 LLDTASALY XLXXXXXXY 1.0155 *25.0000 80 16 POL 752 LLGCAANW XLXXXXXW 95 19 POL 628 LLGFAAPF XLXXXXXF100 20 ENV 378 LLPIFFCLW XLXXXXXXW 100 20 ENV 378 LLPIFFCLWVYXLXXXXXXXXY 26.0549 * 95 19 NUC 44 LLSFLPSDF XLXXXXXXF 95 19 NUC 44LLSFLPSDFF XLXXXXXXXF 90 18 POL 407 LLSSNLSW XLXXXXXW 95 19 ENV 175LLVLQAGF XLXXXXXF 95 19 ENV 175 LLVLQAGFF XLXXXXXXF 20.0121 100 20 ENV338 LLVPFVQW XLXXXXXW 100 20 ENV 338 LLVPFVQWF XLXXXXXXF 85 17 NUC 100LLWFHISCLTF XLXXXXXXXXF 95 19 NUC 45 LSFLPSDF XSXXXXXF 95 19 NUC 45LSFLPSDFF XSXXXXXXF 20.0123 95 19 POL 415 LSLDVSAAF XSXXXXXXF 95 19 POL415 LSLDVSAAFY XSXXXXXXXY 2.0239 * 4.2000 100 20 ENV 336 LSLLVPFVQWXSXXXXXXXW 100 20 ENV 336 LSLLVPFVQWF XSXXXXXXXXF 95 19 X 53 LSLRGLPVCAFXSXXXXXXXXF 95 19 POL 510 LSPFLLAQF XSXXXXXXF 75 15 ENV 349 LSPTVWLSVIWXSXXXXXXXXW 85 17 POL 742 LSRKYTSF XSXXXXXF 85 17 POL 742 LSRKYTSFPWXSXXXXXXXW 75 15 ENV 16 LSVPNPLGF XSXXXXXXF 75 15 NUC 137 LTFGRETVLEYXTXXXXXXXXY 90 18 ENV 189 LTIPQSLDSW XTXXXXXXXW 90 18 ENV 189LTIPQSLDSWW XTXXXXXXXXW 90 18 POL 404 LTNLLSSNLSW XTXXXXXXXXW 95 19 ENV176 LVLQAGFF XVXXXXXF 100 20 ENV 339 LVPFVQWF XVXXXXXF 100 20 POL 377LWDFSQF XVXXXXXF 85 17 ENV 360 MMWYWGPSLY XMXXXXXXXY 1039.01 * 0.0810 7515 X 103 MSTTDLEAY XSXXXXXXY 2.0126 * 0.8500 75 15 X 103 MSTTDLEAYFXSXXXXXXXF 95 19 POL 42 NLGNLNVSIPW XLXXXXXXXXW 90 18 POL 406 NLLSSNLSWXLXXXXXXW 95 19 POL 45 NLNVSIPW XLXXXXXW 75 15 ENV 15 NLSVPNPLGFXLXXXXXXXF 90 18 POL 738 NSVVLSRKY XSXXXXXXY 2.0123 0.0005 100 20 ENV380 PIFFCLWVY XIXXXXXXY 1.0843 0.0078 100 20 ENV 314 PIPSSWAF XIXXXXXF100 20 POL 124 PLDKGIKPY XLXXXXXXY 1.0174 * 0.0190 100 20 POL 124PLDKGIKPYY XLXXXXXXXY 1.0541 * 0.1600 100 20 ENV 377 PLLPIFFCLWXLXXXXXXXW 95 19 ENV 174 PLLVLQAGF XLXXXXXXF 95 19 ENV 174 PLLVLQAGFFXLXXXXXXXF 80 16 POL 505 PMGVGLSPF XMXXXXXXF 85 17 POL 797 PTTGRTSLYXTXXXXXXY 1.0208 * 0.7700 75 15 ENV 351 PTVWLSVIW XTXXXXXXW 85 17 POL612 PVNRPIDW XVXXXXXW 95 19 POL 685 QVFADATPTG XVXXXXXXXXW 90 18 POL 624RIVGLLGF XIXXXXXF 75 15 POL 106 RLKLIMPARF XLXXXXXXXF 75 15 POL 106RLKLIMPARFY XLXXXXXXXXY 95 19 POL 376 RLVVDFSQF XLXXXXXXF 20.0122 90 18POL 353 RTPARVTGGVF XTXXXXXXXXF 100 20 POL 49 SIPWTHKVGNF XIXXXXXXXXF 9519 ENV 194 SLDSWWTSLNF XLXXXXXXXXF 95 19 POL 416 SLDVSAAF XLXXXXXF 95 19POL 416 SLDVSAAFY XLXXXXXXY 1.0186 * 17.2000 100 20 ENV 337 SLLVPFVQWXLXXXXXXW 100 20 ENV 337 SLLVPFVQWF XLXXXXXXXF 95 19 X 54 SLRGLPVCAFXLXXXXXXXF 20.0259 90 18 X 64 SSAGPCALRF XSXXXXXXXF 26.0374 75 15 X 104STTDLEAY XTXXXXXY 75 15 X 104 STTDLEAYF XTXXXXXXF 75 15 ENV 17 SVPNPLGFXVXXXXXF 90 18 POL 739 SVVLSRKY XVXXXXXY 26.0029 85 17 POL 739SVVLSRKYTSF XVXXXXXXXXF 90 18 ENV 190 TIPQSLDSW XIXXXXXXW 90 18 ENV 190TIPQSLDSWW XIXXXXXXXW 100 20 POL 150 TLWKAGILY XLXXXXXXY 1.0177 * 0.001775 15 X 105 TTDLEAYF XTXXXXXF 85 17 POL 798 TTGRTSLY XTXXXXXY 26.0030 8016 NUC 16 TVQASKLCLGW XVXXXXXXXXW 75 15 ENV 352 TVWLSVIW XVXXXXXW 85 17POL 741 VLSRKYTSF XLXXXXXXF 85 17 POL 741 VLSRKYTSFPW XLXXXXXXXXW 85 17POL 740 VVLSRKYTSF XVXXXXXXXF 20.0261 80 16 POL 759 WILRGTSF XIXXXXXF 8016 POL 759 WILRGTSFVY XIXXXXXXXY 1.0572 0.0023 95 19 NUC 125 WIRTPPAYXIXXXXXY 26.0031 80 16 POL 751 WLLGCAANW XLXXXXXXW 95 19 POL 414WLSLDVSAAF XLXXXXXXXF 95 19 POL 414 WLSLDVSAAFY XLXXXXXXXXY 26.0551 10020 ENV 335 WLSLLVPF XLXXXXXF 100 20 ENV 335 WLSLLVPFVQW XLXXXXXXXXW 8517 NUC 26 WLWGMDIDPY XLXXXXXXXY 1.0774 * 0.0810 95 19 ENV 237 WMCLRRFIIFXMXXXXXXXF 20.0266 85 17 ENV 359 WMMWYWGPS XMXXXXXXXXY 26.0552 * 100 20POL 52 WTHKVGNF XTXXXXXF 100 20 POL 122 YLPLDKGIKPY XLXXXXXXXXY 26.055390 18 NUC 118 YLVSFGVW XLXXXXXW 80 16 POL 493 YSHPIILGF XSXXXXXXF 85 17POL 580 YSLNFMGY XSXXXXXY 26.0032 148 17

TABLE VII HBV A02 SUPER MOTIF (With binding information) Conser- Fre- C-vancy quency Protein Position Sequence P2 term Peptide AA Filed A*0201A*0202 A*0203 A*0206 A*6802 85 17 POL 721 AACFARSRSGA A A 11 85 17 POL431 AAMPHLLV A V 8 80 16 POL 756 AANWILRGT A T 9 95 19 POL 632AAPFTQCGYPA A A 11 95 19 POL 521 AICSVVRRA I A 5.0025 9 0.0001 90 18 NUC58 AILCWGEL I L 8 90 18 NUC 58 AILCWGELM I M 9 95 19 POL 642 ALMPLYACI LI 927.15 9 * 0.5000 0.0340 3.3000 0.2500 0.0470 80 16 ENV 108 AMQWNSTT MT 8 75 15 X 102 AMSTTDLEA M A 3.0051 9 0.0013 95 19 POL 690 ATPTGWGL T L8 80 16 POL 690 ATPTGWGLA T A 9 75 15 POL 690 ATPTGWGLAI T I 10 95 19POL 397 AVPNLQSL V L 8 95 19 POL 397 AVPNLQSLT V T 5.0026 9 0.0001 95 19POL 397 AVPNLQSLTNL V L 11 80 16 POL 755 CAANWILRGT A T 10 95 19 X 61CAFSSAGPCA A A 5.0090 10 0.0001 95 19 X 61 CAFSSAGPCAL A L 11 90 18 X 69CALRFTSA A A 8 100 20 ENV 312 CIPIPSSWA I A 5.0007 9 0.0010 80 16 ENV312 CIPIPSSWAFA I A 11 90 18 POL 533 CLAFSYMDDV L V 1.0559 10 0.0008 9018 POL 533 CLAFSYMDDW L V 11 85 17 NUC 23 CLGWLWGM L M 8 85 17 NUC 23CLGWLWGMDI L I 3.0210 10 0.0093 100 20 ENV 253 CLIFLLVL L L Chisari 80.0002 4.011 100 20 ENV 253 CLIFLLVLL L L 1.0836 9 0.0006 95 19 ENV 239CLRRFIIFL L L 1.0829 9 0.0002 75 15 ENV 239 CLRRFIIFLFI L I Chisari 110.0004 4.055 90 18 NUC 107 CLTFGRET L T 8 90 18 NUC 107 CLTFGRETV L V1.0160 9 0.0001 100 20 ENV 310 CTCIPIPSSWA T A 11 95 19 POL 689DATPTGWGL A L 5.0027 9 0.0001 80 16 POL 689 DATPTGWGLA A A 10 75 15 POL689 DATPTGWGLAI A I 11 90 18 NUC 31 DIDPYKEFGA I A 10 85 17 NUC 29DLLDTASA L A 8 85 17 NUC 29 DLLDTASAL L L 1.0154 9 0.0001 95 19 POL 40DLNLGNLNV L V 927.30 9 0.0004 95 19 POL 40 DLNLGNLNVSI L I 11 80 16 NUC32 DTASALYREA T A 10 80 16 NUC 32 DTASALYREAL T L 11 95 19 X 14 DVLCLRPVV V 8 95 19 X 14 DVLCLRPVGA V A 5.0091 10 0.0001 90 18 POL 541 DVVLGAKSVV V 1.0190 9 0.0003 100 20 POL 17 EAGPLEEEL A L 5.0028 9 0.0001 80 16 X122 ELGEEFL L L 8 90 18 POL 718 ELLAACFA L A 8 75 15 NUC 142 ETVLEYLV TV 8 95 19 POL 687 FADATPTGWGL A L 11 85 17 POL 724 FARSRSGA A A 8 80 16POL 821 FASPLHVA A A 8 95 19 POL 396 FAVPNLQSL A L 9 95 19 POL 396FAVPNLQSLT A T 5.0083 10 0.0003 80 16 ENV 243 FIIFLFIL I L Chisari 80.0006 4.047 80 16 ENV 243 FIIFLFILL I L 1.0830 9 0.0002 80 16 ENV 243FIIFLFILLL I L 1.0894 10 0.0012 80 16 ENV 248 FILLLCLI I I Chisari 80.0003 4.048 80 16 ENV 248 FILLLCLIFL I L 1.0895 10 * 0.0280 80 16 ENV248 FlLLLCLIFLL I L Chisari 11 0.0010 4.049 80 16 ENV 246 FLFILLLCL L L1.0832 9 0.0002 80 16 ENV 246 FLFILLLCLI L I 3.0206 10 0.0013 75 15 ENV171 FLGPLLVL L L 8 75 15 ENV 171 FLGPLLVLQA L A 3.0205 10 * 0.0190 95 19POL 513 FLLAQFTSA L A 1069.07 9 * 0.2400 95 19 POL 513 FLLAQFTSAI L I1147.13 10 * 0.2100 0.0320 7.0000 0.1100 0.0880 95 19 POL 562 FLLSLGIHLL L 927.11 9 * 0.6500 0.0010 0.0100 0.1100 0.0035 80 16 ENV 183 FLLTRILTL T 8 80 16 ENV 183 FLLTRILTI L I 777.03 9 * 0.5100 0.0430 8.0000 0.20000.0010 95 19 ENV 256 FLLVLLDYQGM L M 11 100 20 POL 363 FLVDKNPHNT L T5.0084 10 0.0012 95 19 POL 656 FTFSPTYKA T A 1147.15 9 * 0.0056 0.01500.0031 0.8000 7.3000 95 19 POL 656 FTFSPTYKAFL T L 11 95 19 POL 59FTGLYSST T T 8 90 18 POL 59 FTGLYSSTV T V 20.0118 9 0.0005 95 19 POL 635FTQCGYPA T A 8 95 19 POL 635 FTQCGYPAL T L 5.0031 9 0.0009 95 19 POL 635FTQCGYPALM T M 5.0085 10 0.0024 95 19 POL 518 FTSAICSV T V 8 95 19 POL518 FTSAICSVV T V 5.0032 9 0.0090 95 19 ENV 346 FVGLSPTV V V 8 95 19 ENV346 FVGLSPTVWL V L 1.0931 10 0.0008 90 18 X 132 FVLGGCRHKL V L Chisari10 0.0030 4.114 90 18 X 132 FVLGGCRHKLV V V 11 95 19 ENV 342 FVQWFVGL VL 8 95 19 ENV 342 FVQWFVGLSPT V T 11 90 18 POL 766 FVYVPSAL V L 8 90 18POL 766 FVYVPSALNPA V A 11 95 19 X 50 GAHLSLRGL A L 5.0040 9 0.0001 9018 X 50 GAHLSLRGLPV A V 11 85 17 POL 545 GAKSVQHL A L 8 85 17 POL 545GAKSVQHLESL A L 11 75 15 POL 567 GIHLNPNKT I T 9 90 18 POL 155 GILYKRETI T 8 90 18 POL 155 GILYKRETT I T 9 85 17 POL 682 GLCQVFADA L A 1142.049 * 0.0024 85 17 POL 682 GLCQVFADAT L T 10 95 19 POL 627 GLLGFAAPFT L T5.0086 10 0.0049 85 17 ENV 62 GLLGWSPQA L A 1142.07 9 * 0.4000 0.00030.0350 0.2800 0.0005 95 19 X 57 GLPVCAFSSA L A 5.0092 10 0.0008 95 19POL 509 GLSPFLLA L A 8 95 19 POL 509 GLSPFLLAQFT L T 11 100 20 ENV 348GLSPTVWL L L Chisari 8 0.0036 4.012 75 15 ENV 348 GLSPTVWLSV L V 1.051810 * 0.2800 75 15 ENV 348 GLSPTVWLSVI L I Chisari 11 0.0036 4.031 90 18ENV 265 GMLPVCPL M L 8 90 18 POL 735 GTDNSVVL T L 8 75 15 ENV 13GTNLSVPNPL T L 10 80 16 POL 763 GTSFVYVPSA T A 10 80 16 POL 763GTSFVYVPSAL T L 11 80 16 POL 507 GVGLSPFL V L 8 80 16 POL 507 GVGLSPFLLV L Chisari 9 0.0002 4.082 80 16 POL 507 GVGLSPFLLA V A 10 95 19 NUC 123GVWIRTPPA V A 3.0040 9 0.0030 90 18 NUC 104 HISCLTFGRET I T 11 80 16 POL435 HLLVGSSGL L L 927.43 9 0.0031 90 18 X 52 HLSLRGLPV L V 927.02 90.0014 90 18 X 52 HLSLRGLPVCA L A 11 80 16 POL 491 HLYSHPII L I 17.02568 80 16 POL 491 HLYSHPIIL L L 927.47 9 * 0.2200 0.0003 0.9300 0.17000.0530 85 17 POL 715 HTAELLAA T A 8 85 17 POL 715 HTAELLAACFA T A 11 10020 NUC 52 HTALRQAI T I 8 95 19 NUC 52 HTALRQAIL T L 5.0021 9 0.0001 10020 POL 149 HTLWKAGI T I 8 100 20 POL 149 HTLWKAGIL T L 5.0033 9 0.000180 16 ENV 244 IIFLFILL I L Chisari 8 0.0004 4.051 80 16 ENV 244IIFLFILLL I L 1.0831 9 0.0002 80 16 ENV 244 IIFLFILLLCL I L Chisari 110.0002 4.052 80 16 POL 497 IILGFRKI I I 8 80 16 POL 497 IILGFRKIPM I M10 90 18 NUC 59 ILCWGELM L M 8 80 16 POL 498 ILGFRKIPM L M 3.0016 90.0002 100 20 ENV 249 ILLLCLIFL L L 1137.04 9 * 0.0015 100 20 ENV 249ILLLCLIFLL L L 1069.08 10 * 0.0190 0.0001 0.0002 0.1300 0.0015 100 20ENV 249 ILLLCLIFLLV L V Chisari 11 0.0056 4.013 80 16 POL 760 ILRGTSFV LV 8 80 16 POL 760 ILRGTSFVYV L V 1.0573 10 * 0.0160 100 20 NUC 139ILSTLPET L T 8 100 20 NUC 139 ILSTLPETT L T 5.0022 9 0.0001 100 20 NUC139 ILSTLPETTV L V 1069.14 10 * 0.0210 0.0085 0.0770 0.3100 0.0067 10020 NUC 139 ILSTLPETTVV L V 11 95 19 ENV 188 ILTIPQSL L L 8 90 18 POL 156ILYKRETT L T 8 90 18 POL 625 IVGLLGFA V A 8 90 18 POL 625 IVGLLGFAA V A3.0041 9 0.0009 90 18 POL 153 KAGILYKRET A T 10 90 18 POL 153KAGILYKRETT A T 11 80 16 POL 503 KIPMGVGL I L 8 85 17 NUC 21 KLCLGWLWGML M 1142.02 10 * 0.0001 95 19 POL 489 KLHLYSHPI L I 927.46 9 * 0.06900.0340 2.7000 0.5900 0.0015 80 16 POL 489 KLHLYSHPII L I 10 80 16 POL489 KLHLYSHPIIL L L 11 80 16 POL 610 KLPVNRPI L I 8 95 19 POL 574KTKRWGYSL T L 5.0034 9 0.0001 85 17 POL 620 KVCQRIVGL V L 1.0198 90.0003 85 17 POL 620 KVCQRIVGLL V L 1.0567 10 0.0001 95 19 POL 55KVGNFTGL V L 17.0116 8 85 17 X 91 KVLHKRTL V L 8 85 17 X 91 KVLHKRTLGL VL Chisari 10 0.0004 4.115 90 18 POL 534 LAFSYMDDV A V 20.0119 9 0.000290 18 POL 534 LAFSYMDDVV A V 20.0257 10 0.0003 90 18 POL 534 LAFSYMDDVVLA L 11 95 19 POL 515 LAQFTSAI A I 8 95 19 POL 515 LAQFTSAICSV A V 11 10020 ENV 254 LIFLLVLL I L Chisari 8 0.0025 4.014 95 19 POL 514 LLAQFTSA LA 8 95 19 POL 514 LLAQFTSAI L I 1069.05 9 * 0.1000 0.2700 3.7000 0.26000.7900 100 20 ENV 251 LLCLIFLL L L Chisari 8 0.0004 4.015 100 20 ENV 251LLCLIFLLV L V 1137.03 9 * 0.0048 100 20 ENV 251 LLCLIFLLVL L L 1.0898 100.0075 100 20 ENV 251 LLCLIFLLVLL L L Chisari 11 0.0013 4.016 85 17 NUC30 LLDTASAL L L 8 95 19 ENV 260 LLDYQGML L L Chisari 8 0.0004 4.021 9018 ENV 260 LLDYQGMLPV L V 1137.02 10 * 0.0980 0.0001 0.0200 0.67000.0009 80 16 POL 752 LLGCAANWI L I 927.22 9 0.0011 80 16 POL 752LLGCAANWIL L L 1.0912 10 * 0.0140 95 19 POL 628 LLGFAAPFT L T 5.0035 90.0008 85 17 ENV 63 LLGWSPQA L A 8 75 15 ENV 63 LLGWSPQAQGI L I 11 10020 ENV 250 LLLCLIFL L L Chisari 8 0.0006 4.017 100 20 ENV 250 LLLCLIFLLL L 1090.05 9 * 0.0065 100 20 ENV 250 LLLCLIFLLV L V 1137.01 10 * 0.0036100 20 ENV 250 LLLCLIFLLVL L L ChisaRi 11 0.0005 4.018 100 20 ENV 378LLPIFFCL L L Chisari 8 0.0055 4.019 100 20 ENV 378 LLPIFFCLWV L V1069.10 10 * 0.0320 0.0008 0.0150 0.8000 0.0005 95 19 POL 563 LLSLGIHL LL 8 90 18 POL 407 LLSSNLSWL L L 927.41 9 * 0.0110 0.0780 3.9000 0.27000.0100 90 18 POL 407 LLSSNLSWLSL L L 11 80 16 ENV 184 LLTRILTI L IChisari 8 0.0026 4.053 80 16 POL 436 LLVGSSGL L L 8 95 19 ENV 257LLVLLDYQGM L M 3.0207 10 0.0050 95 19 ENV 257 LLVLLDYQGML L L 11 90 18ENV 175 LLVLQAGFFL L L 1090.06 10 * 0.0310 0.0037 0.0045 0.1500 0.011090 18 ENV 175 LLVLQAGFFLL L L Chisari 11 0.0074 4.028 95 19 ENV 338LLVPFVQWFV L V 1069.06 10 * 0.6700 0.3800 1.7000 0.2900 0.1400 90 18 NUC100 LLWFHISCL L L 1142.01 9 * 0.0130 0.0002 0.0420 0.3100 0.0098 85 17NUC 100 LLWFHISCLT L T 10 95 19 POL 643 LMPLYACI M I 17.0130 8 95 19 NUC108 LTFGRETV T V 8 75 15 NUC 137 LTFGRETVL T L 9 90 18 POL 404 LTNLLSSNLT L 9 80 16 ENV 185 LTRILTIPQSL T L 11 85 17 POL 99 LTVNEKRRL T L 9 10020 POL 364 LVDKNPHNT V T 5.0036 9 0.0001 95 19 ENV 258 LVLLDYQGM V M3.0034 9 0.0001 95 19 ENV 258 LVLLDYQGML V L 1.0515 10 0.0001 90 18 ENV176 LVLQAGFFL V L 1.0827 9 0.0096 90 18 ENV 176 LVLQAGFFLL V L 1132.1710 * 0.0022 90 18 ENV 176 LVLQAGFFLLT V T 11 95 19 ENV 339 LVPFVQWFV V V1132.01 9 * 0.0420 0.0150 0.0048 0.7900 2.8000 95 19 ENV 339 LVPFVQWFVGLV L 11 90 18 NUC 119 LVSFGVWI V I Chisari 8 0.0004 4.078 90 18 NUC 119LVSFGVWIRT V T 10 85 17 ENV 360 MMWYWGPSL M L 1039.03 9 * 0.6400 100 20NUC 136 NAPILSTL A L 8 100 20 NUC 136 NAPILSTLPET A T 11 95 19 POL 42NLGNLNVSI L I 3.0008 9 0.0047 90 18 POL 406 NLLSSNLSWL L L 1.0549 100.0016 95 19 POL 45 NLNVSIPWT L T 5.0037 9 0.0005 100 20 POL 400NLQSLTNL L L 8 100 20 POL 400 NLQSLTNLL L L 927.40 9 0.0047 75 15 ENV 15NLSVPNPL L L 8 90 18 POL 411 NLSWLSLDV L V 927.42 9 * 0.0650 0.00510.6400 0.1600 0.0990 90 18 POL 411 NLSWLSLDVSA L A 11 100 20 POL 47NVSIPWTHKV V V 1.0532 10 0.0001 100 20 POL 430 PAAMPHLL A L 8 85 17 POL430 PAAMPHLLV A V 9 90 18 POL 775 PADDPSRGRL A L 10 90 18 ENV 131PAGGSSSGT A T 9 90 18 ENV 131 PAGGSSSGTV A V 10 95 19 POL 641 PALMPLYA AA 8 95 19 POL 641 PALMPLYACI A I 5.0087 10 0.0001 75 15 X 145 PAPCNFFT AT 8 75 15 X 145 PAPCNFFTSA A A 10 80 16 X 11 PARDVLCL A L 8 75 15 X 11PARDVLCLRPV A V 11 90 18 POL 355 PARVTGGV A V 8 90 18 POL 355 PARVTGGVFLA L 10 90 18 POL 355 PARVTGGVFLV A V 11 95 19 NUC 130 PAYRPPNA A A 8 9519 NUC 130 PAYRPPNAPI A I 5.0081 10 0.0001 95 19 NUC 130 PAYRPPNAPIL A L11 85 17 POL 616 PIDWKVCQRI I I Chisari 10 0.0001 4.091 85 17 POL 616PIDWKVCQRIV I V 11 100 20 ENV 380 PIFFCLWV I V 8 100 20 ENV 380PIFFCLWVYI I I Chisari 10 0.0004 3.074 85 17 POL 713 PIHTAELL I L 8 8517 POL 713 PIHTAELLA I A 9 85 17 POL 713 PIHTAELLAA I A 10 80 16 POL 496PIILGFRKI I I 927.48 9 0.0001 80 16 POL 496 PIILGFRKIPM I M 11 100 20NUC 138 PILSTLPET I T 5.0023 9 0.0001 100 20 NUC 138 PILSTLPETT I T5.0082 10 0.0001 100 20 NUC 138 PILSTLPETTV I V Chisari 11 0.0001 5.12580 16 ENV 314 PIPSSWAFA I A 9 95 19 POL 20 PLEEELPRL L L 927.29 9 0.000390 18 POL 20 PLEEELPRLA L A 3.0225 10 0.0001 95 19 ENV 10 PLGFFPDHQL L L1.0511 10 0.0002 100 20 POL 427 PLHPAAMPHL L L 1.0550 10 0.0001 100 20POL 427 PLHPAAMPHLL L L 11 100 20 ENV 377 PLLPIFFCL L L 1069.13 9 *0.0650 0.0001 0.0018 0.1100 0.0047 100 20 ENV 377 PLLPIFFCLWV L V 11 9018 ENV 174 PLLVLQAGFFL L L Chisari 11 0.0008 4.029 80 16 POL 711PLPIHTAEL L L 927.19 9 0.0004 80 16 POL 711 PLPIHTAELL L L 1.0569 100.0001 80 16 POL 711 PLPIHTAELLA L A 11 75 15 POL 2 PLSYQHFRKL L L1.0527 10 0.0001 75 15 POL 2 PLSYQHFRKLL L L 11 85 17 POL 98 PLTVNEKRRLL L 1.0536 10 0.0001 80 16 POL 505 PMGVGLSPFL M L 1.0557 10 0.0001 80 16POL 505 PMGVGLSPFLL M L 11 75 15 POL 692 PTGWGLAI T I 8 80 16 ENV 219PTSNHSPT T T 8 85 17 POL 797 PTTGRTSL T L 8 85 17 POL 797 PTTGRTSLYA T A10 80 16 NUC 15 PTVQASKL T L 8 80 16 NUC 15 PTVQASKLCL T L 10 75 15 ENV351 PTVWLSVI T I 8 75 15 ENV 351 PTVWLSVIWM T M 10 95 19 X 59 PVCAFSSA VA 8 85 17 POL 612 PVNRPIDWKV V V 1.0566 10 0.0002 95 19 POL 654 QAFTFSPTA T 8 95 19 POL 654 QAFTFSPTYKA A A 11 95 19 ENV 179 QAGFFLLT A T 8 8016 ENV 179 QAGFFLLTRI A I 10 80 16 ENV 179 QAGFFLLTRIL A L 11 90 18 NUC57 QAILCWGEL A L 9 90 18 NUC 57 QAILCWGELM A M 10 95 19 ENV 107 QAMQWNSTA T 8 80 16 ENV 107 QAMQWNSTT A T 9 80 16 NUC 18 QASKLCLGWL A L 10 80 16X 8 QLDPARDV L V Chisari 8 0.0001 4.116 80 16 X 8 QLDPARDVL L L 927.01 90.0001 80 16 X 8 QLDPARDVLCL L L Chisari 11 0.0001 4.073 90 18 NUC 99QLLWFHISCL L L 1142.03 10 * 0.0060 85 17 NUC 99 QLLWFHISCLT L T 11 95 19POL 685 QVFADATPT V T 5.0038 9 0.0001 95 19 POL 528 RAFPHCLA A A 8 80 16ENV 187 RILTIPQSL I L Chisari 9 0.0010 4.054 90 16 POL 624 RIVGLLGFA I A9 90 18 POL 624 RIVGLLGFAA I A 10 75 15 POL 106 RLKLIMPA L A 8 90 18 POL353 RTPARVTGGV T V 10 95 19 NUC 127 RTPPAYRPPNA T A 11 95 19 POL 36RVAEDLNL V L 8 90 18 POL 36 RVAEDLNLGNL V L 11 80 16 POL 818 RVHFASPL VL 8 75 15 POL 818 RVHFASPLHV V V 1.0576 10 0.0001 75 15 POL 818RVHFASPLHVA V A 11 100 20 POL 357 RVTGGVFL V L 8 100 20 POL 357RVTGGVFLV V V 1.0181 9 0.0041 90 18 X 65 SAGPCALRFT A T 10 95 19 POL 520SAICSVVRRA A A 5.0088 10 0.0001 90 18 NUC 35 SALYREAL A L 8 100 20 POL49 SIPWTHKV I V 8 95 19 ENV 194 SLDSWWTSL L L F126.64 9 75 15 POL 565SLGIHLNPNKT L T 11 95 19 ENV 337 SLLVPFVQWFV L V 11 75 15 POL 581SLNFMGYV L V 8 75 15 POL 581 SLNFMGYVI L I 927.12 9 0.0038 95 19 X 54SLRGLPVCA L A 3.0030 9 0.0007 90 18 POL 403 SLTNLLSSNL L L 1.0548 100.0014 75 15 ENV 280 STGPCKTCT T T 9 100 20 NUC 141 STLPETTV T V 8 10020 NUC 141 STLPETTVV T V 5.0024 9 0.0019 80 16 ENV 85 STNRQSGRQPT T T 1185 17 POL 548 SVQHLESL V L 8 80 16 ENV 330 SVRFSWLSL V L Chisari 90.0001 4.025 80 16 ENV 330 SVRFSWLSLL V L Chisari 10 0.0004 4.026 80 16ENV 330 SVRFSWLSLLV V V 11 90 18 POL 739 SVVLSRKYT V T 9 95 19 POL 524SVVRRAFPHCL V L 11 85 17 POL 716 TAELLAACFA A A 10 95 19 NUC 53 TALRQAILA L 8 80 16 NUC 33 TASALYREA A A 9 80 16 NUC 33 TASALYREAL A L 10 90 18ENV 190 TIPQSLDSWWT I T 11 100 20 NUC 142 TLPETTVV L V 8 100 20 POL 150TLWKAGIL L L 8 85 17 POL 798 TTGRTSLYA T A 9 75 15 ENV 278 TTSTGPCKT T T9 75 15 ENV 278 TTSTGPCKTCT T T 11 85 17 POL 100 TVNEKRRL V L 8 80 18NUC 16 TVQASKLCL V L 1.0365 9 0.0002 75 15 ENV 352 TVWLSVIWM V M 3.00359 0.0002 95 19 POL 37 VAEDLNLGNL A L 5.0089 10 0.0001 95 19 X 15VLCLRPVGA L A 3.0028 9 0.0014 85 17 POL 543 VLGAKSVCHL L L 1.0560 100.0001 90 18 X 133 VLGGCRHKL L L 927.08 9 0.0009 90 18 X 133 VLGGCRHKLVL V 1.0589 10 0.0001 85 17 X 92 VLHKRTLGL L L 927.03 9 0.0012 95 19 ENV259 VLLDYQGM L M 17.0107 8 95 19 ENV 259 VLLDYQGML L L 1069.09 9 *0.0440 0.0001 0.0210 0.9000 0.0002 90 18 ENV 259 VLLDYQGMLPV L V 1147.1411 * 0.5800 0.2200 4.9000 0.3400 0.0170 95 19 ENV 177 VLQAGFFL L LChisari 8 0.0019 4.027 95 19 ENV 177 VLQAGFFLL L L 1013.14 9 * 0.0660 9519 ENV 177 VLQAGFFLLT L T 5.0066 10 0.0011 100 20 POL 358 VTGGVFLV T V 890 18 POL 542 VVLGAKSV V V 8 90 16 POL 542 VVLGAKSVQHL V L 11 90 18 POL740 VVLSRKYT V T 8 95 19 POL 525 VVRRAFPHCL V L 2.0217 10 0.0003 95 19POL 525 VVRRAFPHCLA V A 11 80 16 POL 759 WILRGTSFV I V 927.24 9 * 0.027080 16 POL 759 WILRGTSFVYV I V 11 80 16 POL 751 WLLGCAANWI L I Chisari 100.0053 4.101 80 16 POL 751 WLLGCAANWIL L L 11 100 20 POL 414 WLSLDVSA LA 8 95 19 POL 414 WLSLDVSAA L A 3.0023 9 0.0059 100 20 ENV 335 WLSLLVPFVL V 1013.0102 9 * 1.1000 0.0380 7.2000 0.3600 0.0310 95 19 ENV 237WMCLRRFI M I 8 95 19 ENV 237 WMCLRRFII M I 1147.10 9 * 0.0005 95 19 ENV237 WMCLRRFIIFL M L Chisari 11 0.0019 4.024 85 17 ENV 359 WMMWYWGPSL M L1137.05 10 * 0.0009 100 20 POL 52 WTHKVGNFT T T 5.0039 9 0.0001 95 19POL 52 WTHKVGNFTGL T L 11 100 20 POL 147 YLHTLWKA L A 8 100 20 POL 147YLHTLWKAGI L I 1069.11 10 * 0.0160 0.0005 0.5600 0.1000 0.0320 100 20POL 147 YLHTLWKAGIL L L 11 100 20 POL 122 YLPLDKGI L I 8 90 18 NUC 118YLVSFGVWI L I 1090.12 9 * 0.3800 90 18 NUC 118 YLVSFGVWIRT L T 11 90 18POL 538 YMDDVVLGA M A 1090.14 9 * 0.0250 0.0001 0.0024 0.1000 0.0002 8517 POL 746 YTSFPWLL T L 8 75 15 POL 746 YTSFPWLLGCA T A 11 90 18 POL 768YVPSALNPA V A 3.0042 9 0.0039 388 45

TABLE VIII HBV A03 SUPER MOTIF (With binding information) Con- ser- Fre-Pro- Posi- C- vancy quency tein tion Sequence P2 term Peptide AA FiledA*0301 A*1101 A*3101 A*3301 A*6801 85 17 POL 721 AACFARSR A R 26.0003 80.0004 0.0003 0.0056 0.0035 0.0014 95 19 POL 521 AICSVVRR I R 26.0004 8−0.0002 0.0003 0.0014 −0.0009 0.0006 90 18 POL 772 ALNPADDPSR L R 1.109010 0.0003 0.0001 85 17 X 70 ALRFTSAR L R 26.0005 8 0.0047 0.0009 0.04500.0230 0.0004 80 16 POL 822 ASPLHVAWR S R 9 75 15 ENV 84 ASTNRQSGR S R1150.60 9 0.0009 0.0002 0.0088 0.0008 0.0001 80 16 POL 755 CAANWILR A R8 85 17 X 69 CALRFTSAR A R 26.0149 9 * 0.0034 0.0230 1.5000 8.00000.7300 90 16 X 17 CLRPVGAESR L R 1.1093 10 0.0011 0.0001 100 20 NUC 48CSPHHTALR S R 5.0055 9 * 0.0029 0.0001 0.0520 0.0250 0.0440 85 17 NUC 29DLLDTASALYR L R 26.0530 11 0.0042 −0.0003 −0.0012 3.7000 0.0410 85 17NUC 32 DTASALYR T R 26.0006 8 0.0004 −0.0002 −0.0009 0.0018 0.0009 95 19POL 17 EAGPLEEELPR A R 26.0531 11 −0.0009 −0.0003 −0.0012 0.0015 0.011090 18 POL 718 ELLAACFAR L R 1.0988 9 0.0002 0.0004 85 17 POL 718ELLAACFARSR L R 26.0532 11 0.0062 0.0016 0.0200 0.2000 0.1600 95 19 NUC174 ETTVVRRR T R 26.0007 8 0.0003 −0.0002 −0.0009 0.1400 0.0027 80 16NUC 174 ETTVVRRRGR T R 1.1073 10 0.0003 0.0001 80 16 POL 821 FASPLHVAWRA R 10 90 18 X 83 FSSAGPCALR S R 10 95 19 POL 856 FTFSPTYK T K 1147.198 * 0.0100 0.0100 0.0023 0.2100 0.0590 95 19 POL 518 FTSAICSVVR T R1.1085 10 0.0003 0.0003 95 19 POL 518 FTSAICSVVRR T R 26.0533 11 0.00650.0092 0.0170 0.0350 1.5000 90 18 X 132 FVLGGCRHK V K 1090.03 9 * 0.04300.0090 75 15 POL 567 GIHLNPNK I K 8 75 15 POL 567 GIHLNPNKTK I K 1.056310 0.0025 0.0011 0.0009 0.0009 0.0003 75 15 POL 567 GIHLNPNKTKR I R 1185 17 NUC 29 GMDIDPYK M K 26.0009 8 0.0006 0.0004 −0.0009 −0.0009 0.000190 16 POL 735 GTDNSVVLSR T R 1090.04 10 * 0.0010 0.0420 0.0030 0.00190.0008 90 16 POL 735 GTDNSVVLSRK T K 1147.17 11 * 0.0140 0.5600 −0.0002−0.0006 0.0001 95 19 NUC 123 GVWIRTPPAYR V R 26.0535 11 * 0.1900 0.17006.8000 0.7300 0.6600 90 18 NUC 104 HISCLTFGR I R 1069.16 9 * 0.01600.0065 75 15 POL 569 HLNPNKTK L K 8 75 15 POL 569 HLNPNKTKR L R 1.0983 90.0025 0.0001 100 20 POL 149 HTLWKAGILYK T K 1147.16 11 * 0.5400 0.44000.0370 0.0720 0.1900 90 18 NUC 105 ISCLTFGR S R 26.0010 8 0.0004 0.00020.0017 −0.0009 0.0017 100 20 POL 153 KAGILYKR A R 26.0011 8 0.0002−0.0002 0.0015 −0.0009 0.0001 80 16 POL 610 KLPVNRPIDWK L K 11 75 15 X130 KVFVLGGCR V R 1.0993 9 * 0.0420 0.0820 0.6000 0.0710 0.0030 85 17POL 720 LAACFARSR A R 20.0129 9 0.0058 0.0065 90 18 POL 719 LLAACFAR L R26.0012 8 0.0024 0.0003 0.0015 0.0029 0.0064 85 17 POL 719 LLAACFARSR LR 10 85 17 NUC 30 LLDTASALYR L R 1.1070 10 0.0050 0.0002 80 16 POL 752LLGCAANWILR L R 11 75 15 POL 564 LSLGIHLNPNK S K 11 95 19 NUC 169LSTLPETTVVR S R 26.0537 11 −0.0009 0.0008 −0.0012 −0.0023 0.0078 75 15POL 3 LSYQHFRK S K 8 85 17 POL 99 LTVNEKRR T R 26.0013 8 −0.0002 −0.0002−0.0009 −0.0009 0.0001 90 18 NUC 119 LVSFGVWIR V R 1090.08 9 * 0.00280.0120 100 20 POL 377 LVVDFSQFSR V R 1069.20 10 * 0.0016 0.3600 0.02600.2300 0.4900 75 15 X 103 MSTTDLEAYFK S K 11 90 18 NUC 75 NLEDPASR L R26.0014 8 −0.0002 −0.0002 −0.0009 −0.0009 0.0001 95 19 POL 45NLNVSIPWTHK L K 26.0538 11 −0.0009 0.0005 −0.0012 −0.0023 0.0019 90 18POL 738 NSVVLSRK S K 26.0015 8 0.0006 0.0010 −0.0009 −0.0009 0.0007 10020 POL 47 NVSIPWTHK V K 1069.16 9 * 0.0620 0.0570 0.0002 0.0100 0.032090 18 POL 775 PADDPSRGR A R 1150.35 9 0.0008 0.0002 0.0004 0.0015 0.000280 16 X 11 PARDVLCLR A R 1150.36 9 0.0002 0.0002 0.0100 0.0180 0.0002 7515 ENV 83 PASTNRQSGR A R 10 90 18 POL 616 PIDWKVCQR I R 1.0985 9 0.00020.0005 80 16 POL 496 PIILGFRK I K 8 95 19 POL 20 PLEEELPR L R 26.0016 80.0002 −0.0002 −0.0009 −0.0009 0.0001 100 20 POL 2 PLSYQHFR L R 26.00178 −0.0002 −0.0002 −0.0009 −0.0009 0.0001 75 15 POL 2 PLSYQHFRK L K1.0161 9 0.0011 0.0031 0.0006 0.0008 0.0002 65 17 POL 98 PLTVNEKR L R26.0018 8 0.0002 −0.0002 −0.0009 −0.0009 0.0001 85 17 POL 98 PLTVNEKRR LR 1.0974 9 0.0008 0.0005 0.0004 0.0027 0.0002 90 18 X 20 PVGAESRGR V R1.0990 9 0.0002 0.0005 0.0004 0.0043 0.0002 85 17 POL 612 PVNRPIDVVK V K1142.06 9 * 0.0310 0.1400 0.0002 0.0006 0.0009 95 19 POL 654 QAFTFSPTYKA K 1090.10 10 * 0.0450 0.5400 0.0010 0.0057 1.2000 80 16 ENV 179QAGFFLLTR A R 9 75 15 NUC 169 QSPRRRRSQSR S R 28.0839 11 80 16 POL 189QSSGILSR S R 8 75 15 POL 106 RLKLIMPAR L R 1.0975 9 * 0.0950 0.00023.1000 0.0490 0.0002 75 15 X 128 RLKVFVLGGCR L R 11 95 19 POL 376RLVVDFSQFSR L R 26.0539 11 * 0.2800 3.8000 2.6000 1.2000 6.1000 95 19NUC 183 RSPRRRTPSPR S R 26.0540 11 −0.0007 −0.0003 0.0190 −0.0023 0.000375 15 NUC 167 RSQSPRRR S R 8 75 15 NUC 167 RSQSPRRRR S R 9 95 19 NUC 188RTPSPRRR T R 26.0019 8 −0.0002 −0.0002 0.0033 0.0014 0.0002 95 19 NUC188 RTPSPRRRR T R 1.0971 9 * 0.0054 0.0005 0.2000 0.0016 0.0003 100 20POL 357 RVTGGVFLVDK V K 1147.18 11 * 0.0190 0.0290 −0.0002 −0.00030.0001 90 18 X 65 SAGPCALR A R 26.0020 8 −0.0002 0.0020 0.0029 0.00240.0360 95 19 POL 520 SAICSVVR A R 26.0021 8 −0.0002 0.0071 0.0280 0.00810.0690 95 19 POL 520 SAICSVVRR A R 1090.11 9 * 0.0058 0.2100 0.01500.0650 0.3800 90 18 POL 771 SALNPADDPSR A R 26.0542 11 −0.0004 −0.0003−0.0012 −0.0023 0.0003 75 15 POL 565 SLGIHLNPNK L K 28.0758 10 * 90 18 X64 SSAGPCALR S R 26.0153 9 * 0.0080 0.1400 0.3300 0.1600 0.7500 95 19NUC 170 STLPETTVVR T R 1069.21 10 * 0.0007 0.0600 0.0080 0.0240 0.025095 19 NUC 170 STLPETTVVRR T R 1083.01 11 0.0150 1.4000 0.1000 0.16000.3100 80 16 ENV 85 STNRQSGR T R 8 75 15 X 104 STTDLEAYFK T K 1.058410 * 0.0066 2.7000 85 17 POL 716 TAELLAACFAR A R 26.0544 11 0.00060.0023 0.0066 0.1600 0.0590 95 19 NUC 171 TLPETTVVR L R 1.0969 9 0.00080.0002 0.0009 0.0024 0.0180 95 19 NUC 171 TLPETTVVRR L R 1069.22 10 *0.0007 0.0230 0.0006 0.0120 0.0440 95 19 NUC 171 TLPETTVVRRR L R 26.054511 * 0.0005 0.0160 0.0061 0.0710 0.6400 100 20 POL 150 TLWKAGILYK L K1069.15 10 * 5.3000 0.3600 0.0051 0.0010 0.0130 100 20 POL 150TLWKAGILYKR L R 26.0546 11 0.0082 0.0095 0.1000 0.1100 0.0640 95 19 POL519 TSAICSVVR S R 5.0057 9 0.0005 0.0008 0.0600 0.0200 0.0820 95 19 POL519 TSAICSVVRR S R 1142.08 10 * 0.0018 0.0006 0.0030 0.0066 0.0048 75 15X 105 TTDLEAYFK T K 1.0215 9 * 0.0006 0.9200 0.0006 0.0012 0.0170 75 15ENV 278 TTSTGPCK T K 8 80 16 NUC 175 TTVVRRRGR T R 1.0970 9 0.00080.0005 0.2500 0.1400 0.0095 80 16 NUC 176 TVVRRRGR V R 3.0324 8 0.00030.0001 80 18 NUC 176 TVVRRRGRSPR V R 28.0837 11 90 18 X 133 VLGGCRHK L K26.0022 8 0.0150 0.0002 −0.0005 −0.0009 0.0001 80 16 ENV 177 VLQAGFFLLTRL R 11 90 18 NUC 120 VSFGVWIR S R 26.0023 8 * 0.0040 0.0290 0.07500.0270 0.0360 100 20 POL 48 VSIPWTHK S K 26.0024 8 * 0.0130 0.01700.0031 0.0013 0.0004 100 20 POL 358 VTGGVFLVDK T K 1069.17 10 * 0.03900.0920 0.0002 0.0006 0.0022 100 20 POL 378 VVDFSQFSR V R 1069.19 9 *0.0015 0.0750 0.0013 0.0170 0.0330 80 16 NUC 177 VVRRRGRSPR V R 1.107410 0.0027 0.0001 80 16 NUC 177 VVRRRGRSPRR V R 28.0838 11 95 19 NUC 125WIRTPPAYR I R 1.0968 9 0.0008 0.0005 90 18 POL 314 WLQFRNSK L K 26.00258 −0.0002 0.0005 0.0020 0.0052 0.0001 85 17 NUC 26 WLWGMDIDPYK L K26.0547 11 0.0030 0.0013 −0.0003 0.0039 0.0490 100 20 POL 122 YLPLDKGIKL K 1.0173 9 0.0001 0.0001 0.0006 0.0008 0.0002 90 18 NUC 118 YLVSFGVWIRL R 1090.13 10 * 0.0005 0.0002 90 18 POL 538 YMDDVVLGAK M K 1090.15 10 *0.0330 0.0043 0.0002 0.0008 0.0001 80 16 POL 493 YSHPIILGFR S R 10 80 16POL 493 YSHPIILGFRK S K 11

TABLE IX HBV A24 SUPER MOTIF (With binding information) Conservancy FreqProtein Position Sequence String Peptide Filed A*2401 95 19 POL 529AFPHCLAF XFXXXXXF 95 19 POL 529 AFPHCLAFSY XFXXXXXXXY 95 19 POL 529AFPHCLAFSYM XFXXXXXXXXM 95 19 X 62 AFSSAGPCAL XFXXXXXXXL 5.0118 0.001290 18 POL 535 AFSYMDDVVL XFXXXXXXXL 13.0130 0.0009 95 19 POL 655AFTFSPTY XFXXXXXY 95 19 POL 655 AFTFSPTYKAF XFXXXXXXXXF 95 19 POL 521AICSVVRRAF XIXXXXXXXF 90 18 NUC 58 AILCWGEL XIXXXXXL 90 18 NUC 58AILCWGELM XIXXXXXXM 95 19 POL 642 ALMPLYACI XLXXXXXXI 3.0012 * 95 19 NUC54 ALRQAILCW XLXXXXXXW 80 16 ENV 108 AMQWNSTTF XMXXXXXXF 95 19 POL 690ATPTGWGL XTXXXXXL 75 15 POL 690 ATPTGWGLAI XTXXXXXXXI 95 19 POL 397AVPNLQSL XVXXXXXL 95 19 POL 397 AVPNLQSLTNL XVXXXXXXXXL 100 20 NUC 131AYRPPNAPI XYXXXXXXI 5.0062 * 0.0260 100 20 NUC 131 AYRPPNAPIL XYXXXXXXXL2.0172 * 0.0220 75 15 POL 607 CFRKLPVNRPI XFXXXXXXXXI 100 20 ENV 312CIPIPSSW XIXXXXXW 100 20 ENV 312 CIPIPSSWAF XIXXXXXXXF 85 17 NUC 23CLGWLWGM XLXXXXXM 85 17 NUC 23 CLGWLWGMDI XLXXXXXXXI 2.0229 100 20 ENV253 CLIFLLVL XLXXXXXL 17.0248 100 20 ENV 253 CLIFLLVLL XLXXXXXXL 1.083695 19 ENV 253 CLIFLLVLLDY XLXXXXXXXXY 26.0548 95 19 ENV 239 CLRRFIIFXLXXXXXF 95 19 ENV 239 CLRRFIIFL XLXXXXXXL 1.0829 75 15 ENV 239CLRRFIIFLF XLXXXXXXXF 75 15 ENV 239 CLRRFIIFLFI XLXXXXXXXXI Chisari4.055 100 20 ENV 310 CTCIPIPSSW XTXXXXXXXW 90 18 NUC 31 DIDPYKEFXIXXXXXF 85 17 NUC 29 DLLDTASAL XLXXXXXXL 1.0154 85 17 NUC 29 DLLDTASALYXLXXXXXXXY 1.0519 * 95 19 POL 40 DLNLGNLNVSI XLXXXXXXXXI 80 16 NUC 32DTASALYREAL XTXXXXXXXXL 85 17 POL 618 DWKVCQRI XWXXXXXI 85 17 POL 618DWKVCQRIVGL XWXXXXXXXXL 90 18 ENV 262 DYQGMLPVCPL XYXXXXXXXXL 3.04410.0002 80 16 X 122 ELGEEIRL XLXXXXXL 95 19 NUC 43 ELLSFLPSDF XLXXXXXXXF95 19 NUC 43 ELLSFLPSDFF XLXXXXXXXXF 90 18 NUC 117 EYLVSFGVW XYXXXXXXW26.0150 90 18 NUC 117 EYLVSFGVWI XYXXXXXXXI 13.0129 * 0.0340 100 20 ENV382 FFCLWVYI XFXXXXXI 80 16 ENV 182 FFLLTRIL XFXXXXXL 80 16 ENV 182FFLLTRILTI XFXXXXXXXI 85 17 ENV 13 FFPDHQLDPAF XFXXXXXXXXF 80 16 ENV 243FIIFLFIL XIXXXXXL 17.0246 80 16 ENV 243 FIIFLFILL XIXXXXXXL 1.0830 80 16ENV 243 FIIFLFILLL XIXXXXXXXL 1.0894 80 16 ENV 248 FILLLCLI XIXXXXXIChisari 4.048 80 16 ENV 248 FILLLCLIF XIXXXXXXF 80 16 ENV 248 FILLLCLIFLXIXXXXXXXL 1.0895 80 16 ENV 248 FILLLCLIFLL XIXXXXXXXXL Chisari 4.049 8016 ENV 246 FLFILLLCL XLXXXXXXL 1.0832 80 16 ENV 246 FLFILLLCLIXLXXXXXXXI 3.0206 80 16 ENV 246 FLFILLLCLIF XLXXXXXXXXF 75 15 ENV 171FLGPLLVL XLXXXXXL 95 19 POL 513 FLLAQFTSAI XLXXXXXXXI 1147.13 * 95 19POL 562 FLLSLGIHL XLXXXXXXL 1.0851 * 80 16 ENV 183 FLLTRILTI XLXXXXXXI3.0005 * 95 19 ENV 256 FLLVLLDY XLXXXXXY 26.0027 95 19 ENV 256FLLVLLDYQGM XLXXXXXXXXM 95 19 POL 656 FTFSPTYKAF XTXXXXXXXF 20.0262 9519 POL 656 FTFSPTYKAFL XTXXXXXXXXL 95 19 POL 635 FTQCGYPAL XTXXXXXXL5.0031 95 19 POL 635 FTQCGYPALM XTXXXXXXXM 5.0085 95 19 ENV 346FVGLSPTVW XVXXXXXXXW 95 19 ENV 346 FVGLSPTVWL XVXXXXXXXL 1.0931 90 18 X132 FVLGGCRHKL XVXXXXXXXL 1.0588 95 19 ENV 342 FVQWFVGL XVXXXXXL17.0109 * 90 18 POL 766 FVYVPSAL XVXXXXXL 17.0260 * 95 19 POL 630GFAAPFTQCGY XFXXXXXXXXY 80 16 ENV 181 GFFLLTRI XFXXXXXI 80 16 ENV 181GFFLLTRIL XFXXXXXXL 80 16 ENV 181 GFFLLTRILTI XFXXXXXXXXI 95 19 ENV 12GFFPDHQL XFXXXXXL 75 15 ENV 170 GFLGPLLVL XFXXXXXXL 80 16 POL 500GFRKIPMGVGL XFXXXXXXXXL 95 19 POL 627 GLLGFAAPF XLXXXXXXF 20.0124 95 19POL 509 GLSPFLLPAQF XLXXXXXXXF 100 20 ENV 348 GLSPTVWL XLXXXXXL Chisari4.012 75 15 ENV 348 GLSPTVWLSVI XLXXXXXXXXI Chisari 4.031 85 17 NUC 29GMDIDPYKEF XMXXXXXXXF 26.0372 90 18 ENV 265 GMLPVCPL XMXXXXXL 90 18 POL735 GTDNSVVL XTXXXXXL 75 15 ENV 13 GTNLSVPNPL XTXXXXXXXL 80 16 POL 763GTSFVYVPSAL XTXXXXXXXXL 80 16 POL 507 GVGLSPFL XVXXXXXL 80 16 POL 507GVGLSPFLL XVXXXXXXL 1.0850 95 19 NUC 123 GVWIRTPPAY XVXXXXXXXV 1.0525 8517 NUC 25 GWLWGMDI XWXXXXXI 85 17 NUC 25 GWLWGMDIDPY XWXXXXXXXXY 85 17ENV 65 GWSPQAQGI XWXXXXXXI 20.0134 0.0024 85 17 ENV 65 GWSPQAQGILXWXXXXXXXL 20.0268 0.0003 95 19 POL 639 GYPALMPL XYXXXXXL 95 19 POL 639GYPALMPLY XYXXXXXXY 2.0060 * 0.0490 95 19 ENV 234 GYRWMCLRRF XYXXXXXXXF2.0171 * 0.0110 95 19 ENV 234 GYRWMCLRRFI XYXXXXXXXXI 85 17 POL 579GYSLNFMGY XYXXXXXXY 2.0058 0.0002 75 15 POL 579 GYSLNFMGYVI XYXXXXXXXXI80 16 POL 820 HFASPLHVAW XFXXXXXXXW 75 15 POL 7 HFRKLLLL XFXXXXXL 80 16POL 435 HLLVGSSGL XLXXXXXXL 1.0187 75 15 POL 569 HLNPNKTKRW XLXXXXXXXW80 16 POL 491 HLYSHPII XLXXXXXI 17.0256 80 16 POL 491 HLYSHPIILXLXXXXXXL 1.0849 * 80 16 POL 491 HLYSHPIILGF XLXXXXXXXXF 85 17 POL 715HTAELLAACF XTXXXXXXXF 100 20 NUC 52 HTALRQAI XTXXXXXI 95 19 NUC 52HTALRQAIL XTXXXXXXL 5.0021 95 19 NUC 52 HTALRQAILCW XTXXXXXXXXW 100 20POL 149 HTLWKAGI XTXXXXXI 100 20 POL 149 HTLWKAGIL XTXXXXXXL 5.0033 10020 POL 149 HTLWKAGILY XTXXXXXXXY 1.0542 * 100 20 POL 146 HYLHTLWKAGIXYXXXXXXXXI 100 20 ENV 381 IFFCLWVY XFXXXXXY 100 20 ENV 381 IFFCLWVYIXFXXXXXXI 5.0058 0.0087 80 16 ENV 245 IFLFILLL XFXXXXXL 80 16 ENV 245IFLFILLLCL XFXXXXXXXL 80 16 ENV 245 IFLFILLLCLI XFXXXXXXXXI 95 19 ENV255 IFLLVLLDY XFXXXXXXY 80 16 ENV 244 IIFLFILL XIXXXXXL 17.0105 80 16ENV 244 IIFLFILLL XIXXXXXXL 1.0831 80 16 ENV 244 IIFLFILLLCL XIXXXXXXXXLChisari 4.052 80 16 POL 497 IILGFRKI XIXXXXXI 17.0124 * 80 16 POL 497IILGFRKIPM XIXXXXXXXM 90 18 NUC 59 ILCWGELM XLXXXXXM 80 16 POL 498ILGFRKIPM XLXXXXXXM 3.0016 100 20 ENV 249 ILLLCLIF XLXXXXXF 100 20 ENV249 ILLLCLIFL XLXXXXXXL 1.0833 * 100 20 ENV 249 ILLLCLIFLL XLXXXXXXXL1.0896 * 80 16 POL 760 ILRGTSFVY XLXXXXXXY 1.0205 * 95 19 ENV 188ILTIPQSL XLXXXXXL 90 18 ENV 188 ILTIPQSLDSW XLXXXXXXXXW 90 18 POL 625IVGLLGFAAPF XVXXXXXXXXF 85 17 ENV 358 IWMMWYWGPS XWXXXXXXXXL 1039.070.0004 95 19 POL 395 KFAVPNLQSL XFXXXXXL 5.0114 0.0020 80 16 POL 503KIPMGVGL XIXXXXXL 80 16 POL 503 KIPMGVGLSPF XIXXXXXXXXF 85 17 NUC 21KLCLGWLW XLXXXXXW 85 17 NUC 21 KLCLGWLWGM XLXXXXXXXM 3.0209 * 95 19 POL489 KLHLYSHPI XLXXXXXXI 3.0009 * 80 16 POL 489 KLHLYSHPII XLXXXXXXXI 8016 POL 489 KLHLYSHPIIL XLXXXXXXXXL 75 15 POL 108 KLIMPARF XLXXXXXF 75 15POL 108 KLIMPARFY XLXXXXXXY 1.0171 80 16 POL 610 KLPVNRPI XLXXXXXI 80 16POL 610 KLPVNRPIDW XLXXXXXXXW 95 19 POL 574 KTKRWGYSL XTXXXXXXL 5.003485 17 POL 574 KTKRWGYSLNF XTXXXXXXXXF 85 17 POL 620 KVCQRIVGL XVXXXXXXL1.0198 85 17 POL 620 KVCQRIVGLL XVXXXXXXXL 1.0567 95 19 POL 55 KVGNFTGLXVXXXXXL 17.0116 95 19 POL 55 KVGNFTGLY XVXXXXXXY 1.0166 * 85 17 X 91KVLHKRTL XVXXXXXL 85 17 X 91 KVLHKRTLGL XVXXXXXXXL 1.0800 100 20 POL 121KYLPLDKGI XYXXXXXXI 5.0063 * 0.0028 85 17 POL 745 KYTSFPWL XYXXXXXL17.0132 85 17 POL 745 KYTSFPWLL XYXXXXXXL 2.0061 * 3.6000 80 16 ENV 247LFILLLCL XFXXXXXL 17.0247 80 16 ENV 247 LFILLLCLI XFXXXXXXI 80 16 ENV247 LFILLLCLIF XFXXXXXXXF 80 16 ENV 247 LFILLLCLIFL XFXXXXXXXXL 100 20ENV 254 LIFLLVLL XIXXXXXL Chisari 4.014 95 19 ENV 254 LIFLLVLLDYXIXXXXXXXY 1.0899 100 20 POL 109 LIMPARFY XIXXXXXY 26.0028 95 19 POL 514LLAQFTSAI XLXXXXXXI 3.0010 * 100 20 ENV 251 LLCLIFLL XLXXXXXL Chisari4.015 100 20 ENV 251 LLCLIFLLVL XLXXXXXXXL 1.0898 100 20 ENV 251LLCLIFLLVLL XLXXXXXXXXL Chisari 4.016 85 17 NUC 30 LLDTASAL XLXXXXXL 8517 NUC 30 LLDTASALY XLXXXXXXY 1.0155 * 95 19 ENV 260 LLDYQGML XLXXXXXLChisari 4.021 80 16 POL 752 LLGCAANW XLXXXXXW 80 16 POL 752 LLGCAANWIXLXXXXXXI 3.0013 80 16 POL 752 LLGCAANWIL XLXXXXXXXL 1.0912 * 95 19 POL628 LLGFAAPF XLXXXXXF 75 15 ENV 63 LLGWSPQAQGI XLXXXXXXXXI 100 20 ENV250 LLLCLIFL XLXXXXXL Chisari 4.017 100 20 ENV 250 LLLCLIFLL XLXXXXXXL1.0834 * 100 20 ENV 250 LLLCLIFLLVL XLXXXXXXXXL Chisari 4.018 100 20 ENV378 LLPIFFCL XLXXXXXL 17.0112 100 20 ENV 378 LLPIFFCLW XLXXXXXXW 100 20ENV 378 LLPIFFCLWVY XLXXXXXXXXY 26.0549 * 95 19 NUC 44 LLSFLPSDFXLXXXXXXF 95 19 NUC 44 LLSFLPSDFF XLXXXXXXXF 95 19 POL 563 LLSLGIHLXLXXXXXL 90 18 POL 407 LLSSNLSW XLXXXXXW 90 18 POL 407 LLSSNLSWLXLXXXXXXL 1.0184 * 90 18 POL 407 LLSSNLSWLSL XLXXXXXXXXL 80 16 ENV 184LLTRILTI XLXXXXXI Chisari 4.053 80 16 POL 436 LLVGSSGL XLXXXXXL 95 19ENV 257 LLVLLDYQGM XLXXXXXXXM 3.0207 95 19 ENV 257 LLVLLDYQGSMLXLXXXXXXXXL 95 19 ENV 175 LLVLQAGF XLXXXXXF 95 19 ENV 175 LLVLQAGFFXLXXXXXXF 20.0121 90 18 ENV 175 LLVLQAGFFL XLXXXXXXXL 1.0892 * 90 18 ENV175 LLVLQAGFFLL XLXXXXXXXXL Chisari 4.028 100 20 ENV 338 LLVPFVQWXLXXXXXW 100 20 ENV 338 LLVPFVQWF XLXXXXXXF 90 18 NUC 100 LLWFHISCLXLXXXXXXL 1.0844 * 85 17 NUC 100 LLWFHISCLTF XLXXXXXXXXF 95 19 POL 643LMPLYACI XMXXXXXI 17.0130 75 15 NUC 137 LTFGRETVL XTXXXXXXL 75 15 NUC137 LTFGRETVLEY XTXXXXXXXXY 90 18 ENV 189 LTIPQSLDSW XTXXXXXXXW 90 18ENV 189 LTIPQSLDSWW XTXXXXXXXXW 90 18 POL 404 LTNLLSSNL XTXXXXXXL 90 18POL 404 LTNLLSSNLSW XTXXXXXXXXW 80 16 ENV 185 LTRILTIPQSL XTXXXXXXXXL 8517 POL 99 LTVNEKRRL XTXXXXXXL 95 19 ENV 258 LVLLDYQGM XVXXXXXXM 3.003495 19 ENV 258 LVLLDYQGML XVXXXXXXXL 1.0515 95 19 ENV 176 LVLQAGFFXVXXXXXF 90 16 ENV 176 LVLQAGFFL XVXXXXXXL 1.0827 90 18 ENV 176LVLQAGFFLL XVXXXXXXXL 1.0893 * 100 20 ENV 339 LVPPVQWF XVXXXXXF 95 19ENV 339 LVPFVQWFVGL XVXXXXXXXXL 90 18 NUC 119 LVSFGVWI XVXXXXXI Chisari4.078 100 20 POL 377 LVVDFSQF XVXXXXXF 90 18 NUC 101 LWFHISCL XWXXXXXL85 17 NUC 101 LWFHISCLTF XWXXXXXXXF 26.0373 85 17 NUC 27 LWGMDIDPYXWXXXXXXY 100 20 POL 151 LWKAGILY XWXXXXXY 80 16 POL 492 LYSHPIILXYXXXXXL 80 16 POL 492 LYSHPIILGF XYXXXXXXXF 2.0161 * 1.1000 85 17 ENV360 MMWYWGPSL XMXXXXXXL 1.0839 * 0.0012 85 17 ENV 360 MMWYWGPSLYXMXXXXXXXY 1039.01 * 0.0001 85 17 ENV 361 MWYWGPSL XWXXXXXL 17.0249 8517 ENV 361 MWYWGPSLY XWXXXXXXY 1039.02 0.0027 95 19 POL 561 NFLLSLGIXFXXXXXI 95 19 POL 561 NFLLSLGIHL XFXXXXXXXL 5.0115 0.0099 95 19 POL 42NLGNLNVSI XLXXXXXXI 3.0008 95 19 POL 42 NLGNLNVSIPW XLXXXXXXXXW 90 18POL 406 NLLSSNLSW XLXXXXXXW 90 18 POL 406 NLLSSNLSWL XLXXXXXXXL 1.054995 19 POL 45 NLNVSIPW XLXXXXXW 100 20 POL 400 NLQSLTNL XLXXXXXL 100 20POL 400 NLQSLTNLL XLXXXXXXL 1.0189 75 15 ENV 15 NLSVPNPL XLXXXXXL 75 15ENV 15 NLSVPNPLGF XLXXXXXXXF 80 16 POL 758 NWILRGTSF XWXXXXXXF 80 16 POL758 NWILRGTSFVY XWXXXXXXXXY 95 19 POL 512 PFLLAQFTSAI XFXXXXXXXXI 95 19POL 634 PFTQCGYPAL XFXXXXXXXL 5.0116 0.0002 95 19 POL 634 PFTQCGYPALMXFXXXXXXXXM 95 19 ENV 341 PFVQWFVGL XFXXXXXXL 5.0059 0.0003 85 17 POL616 PIDWKVCQRI XIXXXXXXXI Chisari 4.091 100 20 ENV 380 PIFFCLWVYXIXXXXXXY 1.0843 100 20 ENV 380 PIFFCLWVYI XIXXXXXXXI 20.0258 85 17 POL713 PIHTAELL XIXXXXXL 80 16 POL 496 PIILGFRKI XIXXXXXXI 927.48 80 15 POL496 PIILGFRKlPM XIXXXXXXXXM 100 20 ENV 314 PIPSSWAF XIXXXXXF 100 20 POL124 PLDKGIKPY XLXXXXXXY 1.0174 * 100 20 POL 124 PLDKGIKPYY XLXXXXXXXY1.0541 * 95 19 POL 20 PLEEELPRL XLXXXXXXL 1.0163 95 19 ENV 10 PLGFFPDHQLXLXXXXXXXL 1.0511 100 20 POL 427 PLHPAAMPHL XLXXXXXXXL 1.0550 100 20 POL427 PLHPAAMPHLL XLXXXXXXXXL 100 20 ENV 377 PLLPIFFCL XLXXXXXXL 1.0842 *100 20 ENV 377 PLLPIFFCLW XLXXXXXXXW 95 19 ENV 174 PLLVLQAGF XLXXXXXXF95 19 ENV 174 PLLVLQAGFF XLXXXXXXXF 90 18 ENV 174 PLLVLQAGFFLXLXXXXXXXXL Chisari 4.029 80 16 POL 711 PLPIHTAEL XLXXXXXXL 1.0201 80 18POL 711 PLPIHTAELL XLXXXXXXXL 1.0569 75 16 POL 2 PLSYQHFRKL XLXXXXXXXL1.0527 75 15 POL 2 PLSYQHFRKLL XLXXXXXXXXL 85 17 POL 98 PLTVNEKRRLXLXXXXXXXL 1.0536 80 16 POL 505 PMGVGLSPF XMXXXXXXF 80 16 POL 505PMGVGLSPFL XMXXXXXXXL 1.0557 80 16 POL 505 PMGVGLSPFLL XMXXXXXXXXL 75 15POL 692 PTGWGLAI XTXXXXXI 85 17 POL 797 PTTGRTSL XTXXXXXL 85 17 POL 797PTTGRTSLY XTXXXXXXY 1.0208 * 80 16 NUC 15 PTVQASKL XTXXXXXL 80 16 NUC 15PTVQASKLCL XTXXXXXXXL 75 15 ENV 361 PTVWLSVI XTXXXXXI 75 15 ENV 351PTVWLSVIW XTXXXXXXW 75 15 ENV 351 PTVWLSVIWM XTXXXXXXXM 85 17 POL 612PVNRPIDW XVXXXXXW 80 16 POL 750 PWLLGCAANW XWXXXXXXXW 80 15 POL 750PWLLGCAANWI XWXXXXXXXXI 100 20 POL 51 PWTHKVGNF XWXXXXXXF 20.0138 *0.0280 80 16 X 8 QLDPARDVL XLXXXXXXL 1.0210 80 16 X 8 QLDPARDVLCLXLXXXXXXXXL Chisari 4.073 90 18 NUC 99 QLLWFHISCL XLXXXXXXXL 1.0908 * 9519 POL 665 QVFADATPTGW XVXXXXXXXXW 95 19 ENV 344 QWFVGLSPTVW XWXXXXXXXX75 15 ENV 242 RFIIFLFI XFXXXXXI 17.0151 75 15 ENV 242 RFIIFLFILXFXXXXXXL 75 15 ENV 242 RFIIFLFILL XFXXXXXXXL 75 15 ENV 242 RFIIFLFILLLXFXXXXXXXXL 100 20 ENV 332 RFSWLSLL XFXXXXXL 100 20 ENV 332 RFSWLSLLVPFXFXXXXXXXXF 80 16 ENV 167 RILTlPQSL XIXXXXXXL 1.0149 90 18 POL 524RIVGLLGF XIXXXXXF 75 15 POL 106 RLKLIMPARF XLXXXXXXXF 75 15 POL 106RLKLIMPARFY XLXXXXXXXXY 95 19 POL 376 RLVVDFSQF XLXXXXXXF 20.0122 90 18POL 355 RTPARVTGGVF XTXXXXXXXXF 95 19 POL 36 RVAEDLNL XVXXXXXL 90 18 POL36 RVAEDLNLGNL XVXXXXXXXXL 80 16 POL 818 RVHFASPL XVXXXXXL 100 20 POL357 RVTGGVFL XVXXXXXL 85 17 POL 577 RWGYSLNF XWXXXXXF 85 17 POL 577RWGYSLNFM XWXXXXXXM 85 17 POL 677 RWGYSLNRMGY XWXXXXXXXXY 95 19 ENV 238RWMCLRRF XWXXXXXF 95 19 ENV 236 RWMCLRRFI XWXXXXXXI 20.0135 * 0.0710 9519 ENV 236 RWMCLRRFII XWXXXXXXXI 20.0269 * 1.1000 95 19 ENV 236RWMCLRRRIF XWXXXXXXXXF 100 20 POL 167 SFCGSPYSW XFXXXXXXW 20.0139 *0.0710 95 19 NUC 46 SFLPSDFF XFXXXXXF 80 16 POL 765 SFVYVPSAL XFXXXXXXL100 20 POL 49 SIPWTHKVGNF XIXXXXXXXXF 95 19 ENV 194 SLDSWWTSL XLXXXXXXL1.0150 95 19 ENV 194 SLDSWWTSLNF XLXXXXXXXXF 95 19 POL 416 SLDVSAAFXLXXXXXF 95 19 POL 416 SLDVSAAFY XLXXXXXXY 1.0186 * 100 20 ENV 337SLLVPFVQW XLXXXXXXW 100 20 ENV 337 SLLVPFVQWF XLXXXXXXXF 75 15 POL 581SLNFMGYVI XLXXXXXXI 3.0011 95 19 X 54 SLRGLPVCAF XLXXXXXXXF 20.0259 9018 POL 403 SLTNLLSSNL XLXXXKXXXL 1.0548 75 15 X 104 STTDLEAY XTXXXXXY 7515 X 104 STTDLEAYF XTXXXXXXF 75 15 ENV 17 SVPNPLGF XVXXXXXF 85 17 POL548 SVQHLESL XVXXXXXL 80 16 ENV 330 SVRFSWLSL XVXXXXXXL 1.0153 80 16 ENV330 SVRFSWLSLL XVXXXXXXXL 1.0517 90 18 POL 739 SVVLSRKY XVXXXXXY 26.002985 17 POL 739 SVVLSRKYTSF XVXXXXXXXXF 95 19 POL 524 SVVRRAFPHCLXVXXXXXXXXL 95 19 POL 413 SWLSLDVSAAF XWXXXXXXXXF 100 20 ENV 334SWLSLLVPF XWXXXXXXF 20.0136 * 0.3900 95 19 POL 392 SWPKFAVPNL XWXXXXXXXL20.0271 * 5.6000 100 20 ENV 197 SWWTSLNF XWXXXXXF 95 19 ENV 197SWWTSLNFL XWXXXXXXL 20.0137 * 0.3800 90 18 POL 537 SYMDDVVL XYXXXXXL 7515 POL 4 SYQHFRKL XYXXXXXL 75 15 POL 4 SYQHFRKLL XYXXXXXXL 2.0042 0.005175 15 POL 4 SVQHFRKLLL XYXXXXXXXL 2.0173 * 0.0660 75 15 POL 4SYQHFRKLLLL XYXXXXXXXXL 75 15 NUC 138 TFGRETVL XFXXXXXL 75 15 NUC 138TFGRETVLEY XFXXXXXXXY 75 15 NUC 138 TFGRETVLEYL XFXXXXXXXXL 95 19 POL657 TFSPTYKAF XFXXXXXXF 5.0064 0.0060 95 19 POL 657 TFSPTYKAFLXFXXXXXXKL 5.0117 0.0043 90 18 ENV 190 TIPQSLDSW XIXXXXXXW 90 18 ENV 190TIPQSLDSWW XIXXXXXXXW 100 20 POL 150 TLWKAGIL XLXXXXXL 100 20 POL 150TLWKAGILY XLXXXXXXY 1.0177 * 75 15 X 105 TTDLEAYF XTXXXXXF 85 17 POL 798TTGRTSLY XTXXXXXY 26.0030 85 17 POL 100 TVNEKRRL XVXXXXXL 80 16 NUC 16TVQASKLCL XVXXXXXXL 1.0365 80 16 NUC 16 TVQASKLCLGW XVXXXXXXXXW 75 15ENV 352 TVWLSVIW XVXXXXXW 75 15 ENV 352 TVWLSVIWM XVXXXXXXM 3.0035 95 19POL 686 VFADATPTGW XFXXXXXXXW 20.0272 * 0.0180 75 15 X 131 VFVLGGCRHKLXFXXXXXXXXL 85 17 POL 543 VLGAKSVQHL XLXXXXXXXL 1.0560 90 18 X 133VLGGCRHKL XLXXXXXXL 1.0220 85 17 X 92 VLHKRTLGL XLXXXXXXL 1.0391 95 19ENV 259 VLLDYQGM XLXXXXXM 17.0107 95 19 ENV 259 VLLDYQGML XLXXXXXXL1.0151 * 95 19 ENV 177 VLQAGFFL XLXXXXXL Chisari 4.027 95 19 ENV 177VLQAGFFLL XLXXXXXXL 1.0828 * 85 17 POL 741 VLSRKYTSF XLXXXXXXF 85 17 POL741 VLSRKYTSFPW XLXXXXXXXXW 80 16 POL 542 VVLGAKSVQHL XVXXXXXXXXL 85 17POL 740 VVLSRKYTSF XVXXXXXXXXF 20.0261 95 19 POL 525 VVRRAFPHCLXVXXXXXXXL 1.0558 95 19 NUC 124 VWIRTPPAY XWXXXXXXY 75 15 ENV 353VWLSVIWM XWXXXXXM 90 18 NUC 102 WFHISCLTF XFXXXXXXF 13.0073 * 0.0300 9519 ENV 345 WFVGLSPTVW XFXXXXXXXW 20.0270 * 0.0120 95 19 ENV 345WFVGLSPTVWL XFXXXXXXXXL 80 16 POL 759 WILRGTSF XIXXXXXF 80 16 POL 759WLRGTSFVY XIXXXXXXXY 1.0572 95 19 NUC 125 WIRTPPAY XIXXXXXY 26.0031 8016 POL 751 WLLGCAANW XLXXXXXXW 80 16 POL 751 WLLGCAANWI XLXXXXXXXIChisari 4.104 80 16 POL 751 WLLGCAANWIL XLXXXXXXXXL 95 19 POL 414WLSLDVSAAF XLXXXXXXXF 95 19 POL 414 WLSLDVSAAFY XLXXXXXXXXY 26.0551 10020 ENV 335 WLSLLVPF XLXXXXXF 100 20 ENV 335 WLSLLVPFVQW XLXXXXXXXXW 8517 NUC 26 WLWGMDIDPY XLXXXXXXXY 1.0774 * 95 19 ENV 237 WMCLRRFI XMXXXXXI95 19 ENV 237 WMCLRRRI XMXXXXXXI 3.0031 * 0.0230 95 19 ENV 237WMCLRRFIIF XMXXXXXXXF 20.0266 0.0013 95 19 ENV 237 WMCLRRFIIFLXMXXXXXXXXL Chisari 4.024 85 17 ENV 359 WMMWYWGPSL XMXXXXXXXL 1.0901 *0.0005 85 17 ENV 359 WMMWYWGPSL XMXXXXXXXXY 26.0552 * 100 20 POL 52WTHKVGNF XTXXXXXF 95 19 POL 52 WTHKVGNFTGL XTXXXXXXXXL 95 19 ENV 198WWTSLNFL XWXXXXXL 95 17 ENV 362 WYWGPSLY XYXXXXXY 3.0362 0.0001 100 20POL 147 YLHTLWKAGI XLXXXXXXXI 7.0066 * 100 20 POL 147 YLHTLWKAGILXLXXXXXXXXL 100 20 POL 122 YLPLDKGI XLXXXXXI 100 20 POL 122 YLPLDKGIKPYXLXXXXXXXXY 26.0553 90 18 NUC 118 YLVSFGVW XLXXXXXW 90 18 NUC 118YLVSFGVWI XLXXXXXI 3.0007 * 85 17 POL 746 YTSFPWLL XTXXXXXL 411 62

TABLE X HBV B07 SUPER MOTIF (With binding information) Con- ser- Fre-Pro- Posi- C- B*3501 vancy quency tein tion Sequence P2 term Peptide AAFiled B*0702 CIR B*5101 B*5301 B*5401 80 16 POL 611 LPVNRPIDW P W 9 8016 POL 611 LPVNRPIDWKV P V 11 80 16 POL 433 MPHLLVGSSGL P L 11 100 20POL 1 MPLSYQHF P F 19.0010 8 * 0.0001 0.0097 0.0120 0.0370 0.0190 75 15POL 1 MPLSYQHFRKL P L 11 90 18 POL 774 NPADDPSRGRL P L 26.0561 11 *0.0120 0.0001 0.0001 −0.0003 0.0001 95 19 ENV 9 NPLGFFPDHQL P L 26.056211 0.0012 0.0021 0.0001 0.0028 0.0001 75 15 POL 571 NPNKTKRW P W 8 75 15POL 571 NPNKTKRWGY P Y 10 95 19 NUC 129 PPAYRPPNA P A 16.0007 9 0.00010.0001 0.0001 0.0002 0.0003 95 19 NUC 129 PPAYRPPNAPI P I 26.0583 110.0003 0.0001 0.0001 −0.0003 0.0001 85 17 ENV 58 PPHGGLLGW P W 20.0141 90.0001 0.0002 0.0001 0.0003 0.0002 100 20 NUC 134 PPNAPILSTL P L 15.021110 0.0001 0.0001 0.0035 0.0001 0.0002 80 16 POL 615 RPIDWKVCQRI P I 11100 20 NUC 133 RPPNAPIL P L 19.0009 8 * 0.0076 0.0001 0.0280 0.00020.0002 100 20 NUC 133 RPPNAPILSTL P L 26.0564 11 * 0.1300 0.0001 0.0018−0.0003 0.0001 100 20 NUC 44 SPEHCSPHHTA P A 26.0565 11 −0.0002 0.00010.0001 −0.0003 0.0011 95 19 POL 511 SPFLLAQF P F 19.0012 8 * 0.55000.0009 0.0180 0.0009 0.0093 95 19 POL 511 SPFLLAQFTSA P A 26.0566 11 *0.0820 0.0001 0.0001 −0.0003 12.0500 100 20 NUC 49 SPHHTALRQA P A16.0178 10 0.0012 0.0001 0.0002 0.0035 100 20 NUC 49 SPHHTALRQAI P I26.0567 11 * 0.5800 0.0001 0.0004 0.0005 0.0002 85 17 ENV 67 SPQAQGIL PL 8 85 17 POL 808 SPSVPSHL P L 8 75 15 ENV 350 SPTVWLSV P V 8 75 15 ENV350 SPTVWLSVI P I 1308.16 9 75 15 ENV 350 SPTVWLSVIW P W 1308.17 10 7515 ENV 350 SPTVWLSVIWM P M 11 95 19 POL 659 SPTYKAFL P L 19.0015 8 *0.3900 0.0001 0.0019 0.0002 0.0002 90 18 POL 354 TPARVTGGV P V 1147.079 * 0.0078 0.0001 0.0013 0.0001 0.0015 90 18 POL 354 TPARVTGGVF P F1147.04 10 * 0.3200 0.1000 0.0001 0.0099 0.0006 90 18 POL 354TPARVTGGVFL P L 26.0568 11 * 0.0950 0.0001 0.0001 0.0005 0.0005 95 19NUC 128 TPPAYRPPNA P A 16.0179 10 * 0.0001 0.0001 0.0002 0.0100 75 15ENV 57 TPPHGGLL P L 8 75 15 ENV 57 TPPHGGLLGW P W 1308.04 10 80 16 POL691 TPTGWGLA P A 8 75 15 POL 691 TPTGWGLAI P I 9 95 19 ENV 340 VPFVQWFVP V 19.0008 8 * 0.0010 0.0001 19.0000 0.0002 0.1100 95 19 ENV 340VPFVQWFVGL P L 15.0213 10 0.0011 0.0001 0.0100 0.0001 0.0025 95 19 POL398 VPNLQSLTNL P L 15.0216 10 0.0006 0.0001 0.0004 0.0001 0.0002 95 19POL 398 VPNLQSLTNLL P L 26.0569 11 0.0004 0.0001 0.0001 −0.0003 0.000290 18 POL 769 VPSALNPA P A 19.0016 8 * 0.0011 0.0001 0.0070 0.00021.0000 95 19 POL 393 WPKFAVPNL P L 15.0035 9 0.0054 0.0002 0.0016 0.00010.0015 95 19 POL 640 YPALMPLY P Y 19.0014 8 * 0.0004 0.2600 0.41000.0450 0.0056 95 19 POL 640 YPALMPLYA P A 1147.08 9 * 0.0180 0.04800.0340 0.0140 16.0000 95 19 POL 640 YPALMPLYACI P I 26.0570 11 0.00400.0001 0.0470 0.0320 0.0700 96 37 75 15 X 146 APCNFFTSA P A 9 95 19 POL633 APFTQCGY P Y 19.0013 8 0.0001 0.0012 0.0019 0.0002 0.0002 95 19 POL633 APFTQCGYPA P A 16.0180 10 * 0.0029 0.0001 0.0002 1.4000 95 19 POL633 APFTQCGYPAL P L 26.0554 11 * 0.2300 0.0010 0.0004 −0.0003 0.0093 10020 ENV 232 CPGYRWMCL P L 1308.21 9 80 16 NUC 14 CPTVQASKL P L 9 80 16NUC 14 CPTVQASKLCL P L 11 80 16 X 10 DPARDVLCL P L 9 80 16 ENV 122DPRVRGLY P Y 8 90 18 POL 778 DPSRGRLGL P L 1147.01 9 * 0.0120 0.00010.0001 0.0001 0.0001 90 18 NUC 33 DPYKEFGA P A 19.0008 8 0.0001 0.00010.0019 0.0002 0.0019 75 15 ENV 130 FPAGGSSSGTV P V 11 90 18 ENV 14FPDHQLDPA P A 1308.23 9 * 85 17 ENV 14 FPDHQLDPAF P F 20.0274 10 0.00020.0016 0.0003 0.0011 0.0021 95 19 POL 530 FPHCLAFSY P Y 1145.08 9 *0.0001 0.5250 0.0665 0.5400 0.0199 95 19 POL 530 FPHCLAFSYM P M 1147.0510 * 0.0990 0.2200 0.0900 0.0790 0.0480 75 15 POL 749 FPWLLGCA P A 8 7515 POL 749 FPWLLGCAA P A 9 75 15 POL 749 FPWLLGCAANW P W 11 90 18 X 67GPCALRFTSA P A 16.0182 10 * 0.0900 0.0001 0.0001 0.0002 0.0035 95 19 POL19 GPLEEELPRL P L 15.0208 10 0.0001 0.0001 0.0002 0.0001 0.0002 90 18POL. 19 GPLEEELPRLA P A 26.0555 11 −0.0002 0.0001 0.0001 −0.0003 0.000195 19 ENV 173 GPLLVLQA P A 19.0003 8 * 0.0003 0.0001 0.0110 0.00020.0065 95 19 ENV 173 GPLLVLQAGF P F 15.0212 10 0.0001 0.0001 0.00020.0001 0.0002 95 19 ENV 173 GPLLVLQAGFF P F 26.0556 11 0.0011 0.00010.0001 0.0008 0.0009 85 17 POL 97 GPLTVNEKRRL P L 26.0557 11 0.00310.0001 0.0001 −0.0003 0.0001 100 20 POL 429 HPAAMPHL P L 19.0011 8 *0.0650 0.0004 0.3100 0.0037 0.0160 100 20 POL 429 HPAAMPHLL P L 1147.029 * 0.0980 0.0270 0.0110 0.0500 0.0120 85 17 POL 429 HPAAMPHLLV P V20.0273 10 * 0.0160 0.0020 0.0078 0.0140 0.0170 80 16 POL 495 HPIILGFRKIP I 10 100 20 ENV 313 IPIPSSWA P A 19.0005 8 * 0.0004 0.0004 0.00190.0002 0.0600 100 20 ENV 313 IPIPSSWAF P F 1145.04 9 * 0.1300 2.76792.3500 0.7450 0.0034 80 16 ENV 313 IPIPSSWAFA P A 16.0177 10 * 0.00130.0024 0.0014 0.4500 80 16 POL 504 IPMGVGLSPF P F 10 80 16 POL 504IPMGVGLSPFL P L 11 90 18 ENV 191 IPQSLDSW P W F126.65 8 90 18 ENV 191IPQSLDSWW P W F126.60 9 * 80 16 ENV 315 IPSSWAFA P A 8 100 20 POL 50IPWTHKVGNF P F 15.0209 10 0.0013 0.0001 0.0007 0.0001 0.0002 100 20 ENV379 LPIFFCLW P W 19.0007 8 * 0.0001 0.0001 0.0360 0.1400 0.0035 100 20ENV 379 LPIFFCLWV P V 1308.22 9 * 100 20 ENV 379 LPIFFCLWVY P Y 15.021510 0.0002 0.0079 0.0002 0.0006 0.0002 100 20 ENV 379 LPIFFCLWVYI P I26.0558 11 0.0002 0.0001 0.0043 0.0139 0.0021 85 17 POL 712 LPIHTAEL P L17.0259 8 85 17 POL 712 LPIHTAELL P L 20.0140 9 * 0.0040 0.0630 0.00520.3100 0.0005 85 17 POL 712 LPIHTAELLA P A 16.0181 10 * 0.0018 0.00110.0016 0.3300 85 17 POL 712 LPIHTAELLAA P A 26.0559 11 0.0090 0.0027−0.0003 0.0120 2.7500 80 16 X 89 LPKVLHKRTL P L 10 100 20 POL 123LPLDKGIKPY P Y 15.0210 10 * 0.0001 0.0290 0.0002 0.0003 0.0002 100 20POL 123 LPLDKGIKPYY P Y 26.0560 11 −0.0002 0.0009 0.0001 0.0007 0.000195 19 X 58 LPVCAFSSA P A 1147.06 9 * 0.0480 0.0710 0.0110 0.0009 19.0000

TABLE XI HBV B27 SUPER MOTIFS Super Source Conservancy Freq ProteinPosition Sequence String Motif Peptide Filed HBV 95 19 X 51 AHLSLRGLXHXXXXXL B27 HBV 85 17 POL 546 AKSVQHLESL XKXXXXXXXL B27 HBV 90 18 POL356 ARVTGGVF XRXXXXXF B27 HBV 90 18 POL 356 ARVTGGVFL XRXXXXXXL B27 HBV95 19 X 48 DHGAHLSL XHXXXXXL B27 HBV 95 19 X 48 DHGAHLSLRGL XHXXXXXXXXLB27 HBV 90 18 ENV 16 DHQLDPAF XHXXXXXF B27 HBV 100 20 POL 126 DKGIKPYYXKXXXXXY B27 HBV 100 20 NUC 46 EHCSPHHTAL XHXXXXXXXL B27 HBV 90 18 NUC103 FHISCLTF XHXXXXXF B27 HBV 80 16 POL 501 FRKIPMGVGL XRXXXXXXXL B27HBV 80 16 POL 608 FRKLPVNRPI XRXXXXXXXI B27 HBV 75 15 NUC 140 GRETVLEYXRXXXXXY B27 HBV 75 15 NUC 140 GRETVLEYL XRXXXXXXL B27 HBV 100 20 NUC 51HHTALRQAI XHXXXXXXI B27 HBV 95 19 NUC 51 HHTALRQAIL XHXXXXXXXL B27 HBV95 19 POL 54 HKVGNFTGL XKXXXXXXL B27 17.0358 HBV 95 19 POL 54 HKVGNFTGLYXKXXXXXXXY B27 HBV 75 15 POL 568 IHLNPNKTKRW XHXXXXXXXXW B27 HBV 85 17POL 714 IHTAELLAACF XHXXXXXXXXF B27 HBV 85 17 POL 576 KRWGYSLNF XRXXXXXFB27 HBV 85 17 POL 576 KRWGYSLNFM XRXXXXXXXM B27 HBV 90 18 X 93 LHKRTLGLXHXXXXXL B27 HBV 95 19 POL 490 LHLYSHPI XHXXXXXI B27 HBV 80 16 POL 490LHLYSHPII XHXXXXXXI B27 HBV 80 16 POL 490 LHLYSHPIIL XHXXXXXXXL B27 HBV100 20 POL 428 LHPAAMPHL XHXXXXXXL B27 HBV 100 20 POL 428 LHPAAMPHLLXHXXXXXXXL B27 HBV 100 20 POL 148 LHTLWKAGI XHXXXXXXI B27 HBV 100 20 POL148 LHTLWKAGIL XHXXXXXXXL B27 HBV 100 20 POL 148 LHTLWKAGILY XHXXXXXXXXYB27 HBV 75 15 POL 107 LKLIMPARF XKXXXXXXF B27 HBV 75 15 POL 107LKLIMPARFY XKXXXXXXXY B27 HBV 95 19 X 55 LRGLPVCAF XRXXXXXXF B27 HBV 8016 POL 761 LRGTSFVY XRXXXXXY B27 HBV 95 19 NUC 55 LRQAILCW XRXXXXXW B27HBV 90 18 NUC 55 LRQAILCWGEL XRXXXXXXXXL B27 HBV 95 19 ENV 240 LRRFIIFLXRXXXXXL B27 HBV 75 15 ENV 240 LRRFIIFLF XRXXXXXXF B27 HBV 75 15 ENV 240LRRFIIFLFI XRXXXXXXXI B27 HBV 75 15 ENV 240 LRRFIIFLFIL XRXXXXXXXXL B27HBV 75 15 POL 573 NKTKRWGY XKXXXXXY B27 HBV 75 15 POL 573 NKTKRWGYSLXKXXXXXXXL B27 HBV 85 17 POL 34 NRRVAEDL XRXXXXXL B27 HBV 85 17 POL 34NRRVAEDLNL XRXXXXXXXL B27 HBV 95 19 POL 531 PHCLAFSY XHXXXXXY B27 HBV 9519 POL 531 PHCLAFSYM XHXXXXXXM B27 HBV 85 17 ENV 59 PHGGLLGW XHXXXXXWB27 HBV 100 20 NUC 50 PHHTALRQAI XHXXXXXXXI B27 HBV 95 19 NUC 50PHHTALRQAIL XHXXXXXXXXL B27 HBV 80 16 POL 434 PHLLVGSSGL XHXXXXXXXL B27HBV 95 19 POL 394 PKFAVPNL XKXXXXXL B27 HBV 95 19 POL 394 PKFAVPNLQSLXKXXXXXXXXL B27 HBV 85 17 X 90 PKVLHKRTL XKXXXXXXL B27 HBV 85 17 X 90PKVLHKRTLGL XKXXXXXXXXL B27 HBV 75 15 POL 6 QHFRKLLL XHXXXXXL B27 HBV 7515 POL 6 QHFRKLLLL XHXXXXXXL B27 HBV 90 18 POL 623 QRIVGLLGF XRXXXXXXFB27 HBV 100 20 POL 145 RHYLHTLW XHXXXXXW B27 HBV 80 16 POL 502 RKIPMGVGLXKXXXXXXL B27 HBV 80 16 POL 609 RKLPVNRPI XKXXXXXXI B27 HBV 80 16 POL609 RKLPVNRPIDW XKXXXXXXXXW B27 HBV 85 17 POL 744 RKYTSFPW XKXXXXXW B27HBV 85 17 POL 744 RKYTSFPWL XKXXXXXXL B27 HBV 85 17 POL 744 RKYTSFPWLLXKXXXXXXXL B27 HBV 95 19 POL 527 RRAFPHCL XRXXXXXL B27 HBV 95 19 POL 527RRAFPHCLAF XRXXXXXXXF B27 HBV 75 15 ENV 241 RRFIIFLF XRXXXXXF B27 HBV 7515 ENV 241 RRFIIFLFI XRXXXXXXI B27 HBV 75 15 ENV 241 RRFIIFLFILXRXXXXXXXL B27 HBV 75 15 ENV 241 RRFIIFLFILL XRXXXXXXXXL B27 HBV 75 15POL 105 RRLKLIMPARF XRXXXXXXXXF B27 HBV 90 16 POL 35 RRVAEDLNL XRXXXXXXLB27 HBV 80 16 POL 494 SHPIILGF XHXXXXXF B27 HBV 80 16 POL 494SHPIILGFRKI XHXXXXXXXXI B27 HBV 90 18 NUC 20 SKLCLGWL XKKXXXXL B27 HBV85 17 NUC 20 SKLCLGWLW XKXXXXXXW B27 HBV 85 17 NUC 20 SKLCLGWLWGMXKXXXXXXXXM B27 HBV 85 17 POL 743 SRKYTSFPW XRXXXXXXW B27 HBV 85 17 POL743 SRKYTSFPWL XRXXXXXXXL B27 HBV 85 17 POL 743 SRKYTSFPWLL XRXXXXXXXXLB27 HBV 95 19 POL 375 SRLVVDFSQF XRXXXXXXXF B27 HBV 80 16 POL 472SRNLYVSL XRXXXXXL B27 17.0123 HBV 95 19 POL 53 THKVGNFTGL XHXXXXXXXL B27HBV 95 19 POL 53 THKVGNFTGLY XHXXXXXXXXY B27 HBV 95 19 POL 575 TKRWGYSLXKXXXXXL B27 HBV 85 17 POL 575 TKRWGYSLNF XKXXXXXXXF B27 HBV 85 17 POL575 TKRWGYSLNFM XKXXXXXXXXM B27 HBV 100 20 POL 120 TKYLPLDKGI XKXXXXXXXIB27 HBV 100 20 POL 144 TRHYLHTL XRXXXXXL B27 HBV 100 20 POL 144TRHYLHTLW XRXXXXXXW B27 HBV 80 16 ENV 186 TRILTIPQSL XRXXXXXXXL B27 HBV80 16 POL 819 VHFASPLHVAW XHXXXXXXXXW B27 HBV 80 16 ENV 331 VRFSWLSLXRXXXXXL B27 HBV 80 16 ENV 331 VRFSWLSLL XRXXXXXXL B27 HBV 95 19 POL 526VRRAFPHCL XRXKXXXXL B27 HBV 95 19 POL 526 VRRAFPHCLAF XRXXXXXXXXF B27HBV 85 17 POL 619 WKVCQRIVGL XKXXXXXXXL B27 HBV 85 17 POL 619WKVCQRIVGLL XKXXXXXXXXL B27 HBV 100 20 NUC 132 YRPPNAPI XRXXXXXI B27 HBV100 20 NUC 132 YRPPNAPIL XRXXXXXXL B27 17.0356 HBV 95 19 ENV 235YRWMCLRRF XRXXXXXXF B27 HBV 95 19 ENV 235 YRWMCLRRFI XRXXXXXXXI B27 HBV95 19 ENV 235 YRWMCLRRFII XRXXXXXXXXI B27 104 0

TABLE XII HBV B44 SUPER MOTIF Source Conservancy Freq Protein PositionSequence String Supermotif Peptide Filed HBV 95 19 POL 688 ADATPTGWXDXXXXXW B44 HBV 95 19 POL 688 ADATPTGWGL XDXXXXXXXL B44 HBV 80 16 POL688 ADATPTGWGL XDXXXXXXXXA B44 HBV 90 18 POL 776 ADDPSRGRL XDXXXXXXL B44HBV 90 18 POL 776 ADDPSRGRLGL XDXXXXXXXXL B44 HBV 95 19 POL 38 AEDLNLGNLXEXXXXXXL B44 17.0357 HBV 95 19 POL 38 AEDLNLGNLNV XEXXXXXXXXV B44 HBV85 17 POL 717 AELLAACF XEXXXXXF B44 HBV 85 17 POL 717 AELLAACFAXEXXXXXXA B44 HBV 90 18 POL 777 DDPSAGRL XDXXXXXL B44 17.0010 HBV 90 18POL 777 DDPSRGRLGL XDXXXXXXXL B44 17.0418 HBV 90 18 POL 540 DDVVLGAKSVXDXXXXXXXV B44 HBV 75 15 POL 18 DEAGPLEEEL XEXXXXXXXL B44 HBV 95 19 POL39 EDUNLGNL XDXXXXXL B44 HBV 95 19 POL 39 EDUNLGNUNV XDXXXXXXXV B44 HBV90 18 POL 22 EEELPRLA XEXXXXXA B44 HBV 80 16 X 121 EELGEEIRL XEXXXXXXLB44 HBV 90 18 NUC 32 IDPYKEFGA XDXXXXXXA B44 HBV 85 17 POL 617 IDWKVCORIXDXXXXXXI B44 HBV 85 17 POL 617 IDWKVCORIV XDXXXXXXXV B44 HBV 100 20 POL125 LDKGIKPY XDXXXXXY B44 HBV 100 20 POL 125 LDKGIKPYY XDXXXXXXY B44 HBV80 16 X 9 LDPARDVL XDXXXXXL B44 17.0012 HBV 80 16 X 9 LDPARDVLCLXDXXXXXXXL B44 17.0419 HBV 95 19 ENV 195 LDSWWTSL XDXXXXXL B44 HBV 95 19ENV 195 LDSWWTSLNF XDXXXXXXXF B44 HBV 90 18 BW 195 LDSWWTSLNFLXDXXXXXXXXL B44 HBV 85 17 NUC 31 LDTASALY XDXXXXXY B44 HBV 80 16 NUC 31LDTASALYREA XDXXXXXXXXA B44 HBV 95 19 POL 417 LDVSAAFY XDXXXXXY B44 HBV90 18 ENV 261 LDYQGMLPV XDXXXXXXV B44 HBV 95 19 POL 21 LEEELPRL XEXXXXXLB44 HBV 90 18 POL 21 LEEELPRLA XEXXXXXXA B44 HBV 90 18 POL 539 MDDVVLGAXDXXXXXA B44 HBV 90 18 POL 539 MDDVVGAKSV XDXXXXXXXXV B44 HBV 90 18 NUC30 MDIDPYKEF XDXXXXXXF B44 HBV 90 18 NUC 30 MDIDPYKEFGA XDXXXXXXXXA B44HBV 95 19 ENV 15 PDHQLDPA XDXXXXXA B44 HBV 90 18 ENV 15 PDHQLDPAFXDXXXXXXF B44 HBV 100 20 NUC 45 PEHCSPHHTA XEXXXXXXXA B44 HBV 100 20 NUC45 PEHCSPHHTAL XEXXXXXXXXL B44 HBV 85 17 NUC 28 RDLLDTASA XDXXXXXXA B44HBV 85 17 NUC 28 RDLLDTASAL XDXXXXXXXL B44 HBV 85 17 NUC 28 RDLLDTASALYXDXXXXXXXXY B44 HBV 95 19 X 13 RDVLCLRPV XDXXXXXXV B44 HBV 95 19 X 13RDVLCLRPVGA XDXXXXXXXXA B44 HBV 75 15 NUC 141 RETVLEYL XEXXXXXL B44 HBV75 15 NUC 141 RETVLEYLV XEXXXXXXV B44 HBV 90 18 POL 736 TDNSVVLSRKYXDXXXXXXXXY B44 HBV 95 19 NUC 42 VELLSFLPSDF XEXXXXXXXXF B44 HBV 80 16 X120 WEELGEEEI XEXXXXXI B44 HBV 80 16 X 120 WEELGEEIRL XEXXXXXXXL B44 520

TABLE XIII HBV B58 SUPER MOTIFS Source Convervancy Freq Protein PositionSequence String Super Motif Peptide Filed HBV 85 17 POL 431 AAMPHLLVXAXXXXXV B58 HBV 95 19 POL 632 AAPFTQCGY XAXXXXXXY B58 HBV 85 17 NUC 34ASALYREAL XSXXXXXXL B58 HBV 100 20 POL 166 ASFCGSPY XSXXXXXY B5826.0026 * HBV 100 20 POL 166 ASFCGSPYSW XSXXXXXXXW B58 HBV 90 18 NUC 19ASKLCLGW XSXXXXXW B58 HBV 90 18 NUC 19 ASKLCLGWL XSXXXXXXL B58 HBV 85 17NUC 19 ASKLCLGWLW XSXXXXXXXW B58 HBV 80 16 POL 822 ASPLHVAW XSXXXXXW B58HBV 80 16 ENV 329 ASVRFSWL XSXXXXXL B58 HBV 80 16 ENV 329 ASVRFSWLSLXSXXXXXXXL B58 HBV 80 16 ENV 329 ASVRFSWLSLL XSXXXXXXXXL B58 HBV 95 19POL 690 ATPTGWGL XTXXXXXL B58 HBV 75 15 POL 690 ATPTGWGLAI XTXXXXXXXIB58 HBV 95 19 X 61 CAFSSAGPCAL XAXXXXXXXXL B58 HBV 100 20 NUC 48CSPHHTAL XSXXXXXL B58 HBV 80 16 POL 471 CSRNLYVSL XSXXXXXXL B58 HBV 9519 POL 523 CSVVRRAF XSXXXXXF B58 HBV 100 20 ENV 310 CTCIPIPSSWXTXXXXXXXW B58 HBV 95 19 POL 689 DATPTGWGL XAXXXXXXL B58 5.0027 HBV 7515 POL 689 DATPTGWGLAI XAXXXXXXXXI B58 HBV 95 19 ENV 196 DSWWTSLNFXSXXXXXXF B58 20.0120 HBV 90 18 ENV 196 DSWWTSLNFL XSXXXXXXXL B58 HBV 8016 NUC 32 DTASALYREAL XTXXXXXXXXL B58 HBV 100 20 POL 17 EAGPLEEELXAXXXXXXL B58 5.0028 HBV 95 19 POL 374 ESRLVVDF XSXXXXXF B58 HBV 95 19POL 374 ESRLVVDFSQF XSXXXXXXXXF B58 HBV 75 15 NUC 142 ETVLEYLV XTXXXXXVB58 HBV 95 19 POL 631 FAAPFTQCGY XAXXXXXXXV B58 20.0254 * HBV 95 19 POL687 FADATPTGW XAXXXXXXW B58 HBV 95 19 POL 687 FADATPTGWGL XAXXXXXXXXLB58 HBV 80 16 POL 821 FASPLHVAW XAXXXXXXW B58 HBV 95 19 POL 396FAVPNLQSL XAXXXXXXL B58 5.0029 * HBV 95 19 POL 658 FSPTYKAF XSXXXXXF B58HBV 95 19 POL 658 FSPTYKAFL XSXXXXXXL B58 HBV 95 19 X 63 FSSAGPCALXSXXXXXXL B58 HBV 90 18 X 63 FSSAGPCALRF XSXXXXXXXXF B58 HBV 100 20 ENV333 FSWLSLLV XSXXXXXV B58 HBV 100 20 ENV 333 FSWLSLLVPF XSXXXXXXXF B5820.0263 HBV 100 20 ENV 333 FSWLSLLVPFV XSXXXXXXXXV B58 HBV 90 18 POL 536FSYMDDVV XSXXXXXV B58 17.0257 HBV 90 18 POL 536 FSYMDDVVL XSXXXXXXL B58HBV 95 19 POL 656 FTFSPTYKAF XTXXXXXXXF B58 20.0262 HBV 95 19 POL 656FTFSPTYKAFL XTXXXXXXXXL B58 HBV 90 18 POL 59 FTGLYSSTV XTXXXXXXV B5820.0118 HBV 95 19 POL 635 FTQCGYPAL XTXXXXXXL B58 5.0031 HBV 95 19 POL635 FTQCGYPALM XTXXXXXXXM B58 5.0085 HBV 95 19 POL 518 FTSAICSV XTXXXXXVB58 HBV 95 19 POL 518 FTSAICSVV XTXXXXXXV B58 5.0032 HBV 95 19 X 50GAHLSLRGL XAXXXXXXL B58 5.0040 HBV 90 18 X 50 GAHLSLRGLPV XAXXXXXXXXVB58 HBV 85 17 POL 545 GAKSVQHL XAXXXXXL B58 HBV 85 17 POL 545GAKSVQHLESL XAXXXXXXXXL B58 HBV 75 15 ENV 134 GSSSGTVNPV XSXXXXXXXV B58HBV 90 18 POL 735 GTDNSVVL XTXXXXXL B58 HBV 75 15 ENV 13 GTNLSVPNPLXTXXXXXXXL B58 HBV 80 16 POL 763 GTSFVYVPSAL XTXXXXXXXXL B58 HBV 85 17POL 715 HTAELLAACF XTXXXXXXXF B58 HBV 100 20 NUC 52 HTALRQAI XTXXXXXIB58 HBV 95 19 NUC 52 HTALRQAIL XTXXXXXXL B58 5.0021 HBV 95 19 NUC 52HTALRQAILCW XTXXXXXXXXW B58 HBV 100 20 POL 149 HTLWKAGI XTXXXXXI B58 HBV100 20 POL 149 HTLWKAGIL XTXXXXXXL B58 5.0033 HBV 100 20 POL 149HTLWKAGILY XTXXXXXXXY B58 1.0542 * HBV 90 18 NUC 105 ISCLTFGRETVXSXXXXXXXXV B58 HBV 85 17 POL 547 KSVQHLESL XSXXXXXXL B58 HBV 95 19 POL574 KTKRWGYSL XTXXXXXXL B58 5.0034 HBV 85 17 POL 574 KTKRWGYSLNFXTXXXXXXXXF B58 HBV 90 18 POL 534 LAFSYMDDV XAXXXXXXV B58 20.0118 HBV 9018 POL 534 LAFSYMDDVV XAXXXXXXXV B58 20.0257 HBV 90 18 POL 534LAFSYMDDVVL XAXXXXXXXXL B58 HBV 95 19 POL 515 LAQFTSAI XAXXXXXI B58 HBV95 19 POL 515 LAQFTSAICSV XAXXXXXXXXV B58 HBV 95 19 NUC 45 LSFLPSDFXSXXXXXF B58 HBV 95 19 NUC 45 LSFLPSDFF XSXXXXXXF B58 20.0123 HBV 95 19POL 415 LSLDVSAAF XSXXXXXXF B58 HBV 95 19 POL 415 LSLDVSAAFY XSXXXXXXXYB58 2.0239 * HBV 100 20 ENV 336 LSLLVPFV XSXXXXXV B58 HBV 100 20 ENV 336LSLLVPFVQW XSXXXXXXXW B58 HBV 100 20 ENV 336 LSLLVPFVQWF XSXXXXXXXXF B58HBV 95 19 X 53 LSLRGLPV XSXXXXXV B58 HBV 95 19 X 53 LSLRGLPVCAFXSXXXXXXXXF B58 HBV 95 19 POL 510 LSPFLLAQF XSXXXXXXF B58 HBV 75 15 ENV349 LSPTVWLSV XSXXXXXXV B58 HBV 75 15 ENV 349 LSPTVWLSVI XSXXXXXXXI B58HBV 75 15 ENV 349 LSPTVWLSVIW XSXXXXXXXXW B58 HBV 85 17 POL 742 LSRKYTSFXSXXXXXF B58 HBV 85 17 POL 742 LSRKYTSFPW XSXXXXXXXW B58 HBV 85 17 POL742 LSRKYTSFPWL XSXXXXXXXXL B58 HBV 90 18 POL 408 LSSNLSWL XSXXXXXL B58HBV 90 18 POL 408 LSSNLSWLSL XSXXXXXXXL B58 HBV 100 20 NUC 140 LSTLPETTVXSXXXXXXV B58 HBV 100 20 NUC 140 LSTLPETTVV XSXXXXXXXV B58 HBV 75 15 ENV16 LSVPNPLGF XSXXXXXXF B58 HBV 100 20 POL 412 LSWLSLDV XSXXXXXV B58 HBV75 15 POL 3 LSYQHFRKL XSXXXXXXL B58 HBV 75 15 POL 3 LSYQHFFRKLLXSXXXXXXXL B58 HBV 75 15 POL 3 LSYQHFRKLLL XSXXXXXXXXL B58 HBV 95 19 NUC108 LTFGRETV XTXXXXXV B58 HBV 75 15 NUC 137 LTFGRETVL XTXXXXXXL B58 HBV75 15 NUC 137 LTFGRETVLEY XTXXXXXXXXY B58 HBV 90 18 ENV 189 LTIPQSLDSWXTXXXXXXXW B58 HBV 90 18 ENV 189 LTIPQSLDSWW XTXXXXXXXXW B58 HBV 90 18POL 404 LTNLLSSNL XTXXXXXXL B58 HBV 90 18 POL 404 LTNLLSSNLSWXTXXXXXXXXW B58 HBV 80 16 ENV 185 LTRILTIPQSL XTXXXXXXXXL B58 HBV 85 17POL 99 LTVNEKRRL XTXXXXXXL B58 HBV 75 15 X 103 MSTTDLEAY XSXXXXXXY B582.0126 * HBV 75 15 X 103 MSTTDLEAYF XSXXXXXXXF B58 HBV 100 20 NUC 136NAPILSTL XAXXXXXL B58 HBV 90 18 POL 738 NSVVLSRKY XSXXXXXXY B58 2.0123HBV 100 20 POL 430 PAAMPHLL XAXXXXXL B58 HBV 85 17 POL 430 PAAMPHLLVXAXXXXXXV B58 HBV 90 18 POL 775 PADDPSRGRL XAXXXXXXXL B58 HBV 90 18 ENV131 PAGGSSSGTV XAXXXXXXXV B58 HBV 95 19 POL 641 PALMPLYACI XAXXXXXXXIB58 5.0087 HBV 80 16 X 11 PARDVLCL XAXXXXXL B58 HBV 75 15 X 11PARDVLCLRPV XAXXXXXXXXV B58 HBV 90 18 POL 355 PARVTGGV XAXXXXXV B58 HBV90 18 POL 355 PARVTGGVF XAXXXXXXF B58 HBV 90 18 POL 355 PARVTGGVFLXAXXXXXXXL B58 HBV 90 18 POL 355 PARVTGGVFLV XAXXXXXXXXV B58 HBV 95 19NUC 130 PAYRPPNAPI XAXXXXXXXI B58 5.0081 HBV 95 19 NUC 130 PAYRPPNAPILXAXXXXXXXXL B58 HBV 90 18 POL 779 PSRGRLGL XSXXXXXL B58 HBV 75 15 POL692 PTGWGLAI XTXXXXXI B58 HBV 85 17 POL 797 PTTGRTSL XTXXXXXL B58 HBV 8517 POL 797 PTTGRTSLY XTXXXXXXY B58 1.0208 * HBV 80 16 NUC 15 PTVQASKLXTXXXXXL B58 HBV 80 16 NUC 15 PTVQASKLCL XTXXXXXXXL B58 HBV 75 15 ENV351 PTVWLSVI XTXXXXXI B58 HBV 75 15 ENV 351 PTVWLSVIW XTXXXXXXW B58 HBV75 15 ENV 351 PTVWLSVIWM XTXXXXXXXM B58 HBV 95 19 POL 654 QAFTFSPTYXAXXXXXXY B58 20.0127 HBV 80 16 ENV 179 QAGFFLLTRI XAXXXXXXXI B58 HBV 8016 ENV 179 QAGFFLLTRIL XAXXXXXXXXL B58 HBV 90 18 NUC 57 QAILCWGELXAXXXXXXL B58 HBV 90 18 NUC 57 QAILCWGELM XAXXXXXXXM B58 HBV 80 16 ENV107 QAMQWNSTTF XAXXXXXXXF B58 HBV 80 16 NUC 18 QASKLCLGW XAXXXXXXW B58HBV 80 16 NUC 18 QASKLCLGWL XAXXXXXXXL B58 HBV 75 15 NUC 18 QASKLCLGWLWXAXXXXXXXXW B58 HBV 90 18 ENV 193 QSLDSWWTSL XSXXXXXXXL B58 F126.63 HBV90 18 POL 402 QSLTNLLSSNL XSXXXXXXXXL B58 HBV 95 19 POL 528 RAFPHCLAFXAXXXXXXF B58 20.0125 HBV 95 19 POL 528 RAFPHCLAFSY XAXXXXXXXXY B5826.0550 * HBV 90 18 POL 353 RTPARVTGGV XTXXXXXXXV B58 HBV 90 18 POL 353RTPARVTGGVF XTXXXXXXXXF B58 HBV 90 18 X 65 SAGPCALRF XAXXXXXXF B5826.0152 HBV 95 19 POL 520 SAICSVVRRAF XAXXXXXXXXF B58 HBV 90 18 NUC 35SALYREAL XAXXXXXL B58 HBV 100 20 POL 165 SASFCGSPY XAXXXXXXY B5820.0117 * HBV 100 20 POL 165 SASFCGSPYSW XAXXXXXXXXW B58 HBV 95 19 X 64SSAGPCAL XSXXXXXL B58 HBV 90 18 X 64 SSAGPCALRF XSXXXXXXXF B58 26.0374HBV 75 15 ENV 136 SSGTVNPV XSXXXXXV B58 HBV 90 18 POL 409 SSNLSWLSLXSXXXXXXL B58 HBV 90 18 POL 409 SSNLSWSLDV XSXXXXXXXXV B58 HBV 75 15 ENV135 SSSGTVNPV XSXXXXXXV B58 HBV 100 20 NUC 141 STLPETTV XTXXXXXV B58 HBV100 20 NUC 141 STLPETTVV XTXXXXXXV B58 5.0024 HBV 75 15 X 104 STTDLEAYXTXXXXXY B58 HBV 75 15 X 104 STTDLEAYF XTXXXXXXF B58 HBV 85 17 POL 716TAELLAACF XAXXXXXXF B58 HBV 95 19 NUC 53 TALRQAIL XAXXXXXL B58 HBV 95 19NUC 53 TALRQAILCW XAXXXXXXXW B58 HBV 80 16 NUC 33 TASALYREAL XAXXXXXXXLB58 HBV 95 19 POL 519 TSAICSVV XSXXXXXV B58 HBV 80 16 POL 764 TSFVYVPSALXSXXXXXXXL B58 HBV 80 16 ENV 168 TSGFLGPL XSXXXXXL B58 HBV 75 15 ENV 168TSGFLGPLL XSXXXXXXL B58 HBV 75 15 ENV 168 TSGFLGPLLV XSXXXXXXXV B58 HBV75 15 ENV 168 TSGFLGPLLVL XSXXXXXXXXL B58 HBV 75 15 X 105 TTDLEAYFXTXXXXXF B58 HBV 85 17 POL 798 TTGRTSLY XTXXXXXY B58 26.0030 HBV 95 19POL 37 VAEDLNLGNL XAXXXXXXXL B58 5.0089 HBV 100 20 POL 48 VSIPWTHKVXSXXXXXXV B58 HBV 95 19 POL 391 VSWPKFAV XSXXXXXV B58 HBV 95 19 POL 391VSWPKFAVPNL XSXXXXXXXXL B58 HBV 100 20 POL 358 VTGGVFLV XTXXXXXV B58 HBV85 17 ENV 66 WSPQAQGI XSXXXXXI B58 HBV 85 17 ENV 66 WSPQAQGIL XSXXXXXXLB58 HBV 100 20 POL 52 WTHKVGNF XTXXXXXF B58 HBV 95 19 POL 52 WTHKVGNFTGLXTXXXXXXXXL B58 HBV 80 16 POL 493 VSHPIILGF XSXXXXXXF B58 HBV 85 17 POL580 VSLNFMGY XSXXXXXY B58 26.0032 HBV 75 15 POL 580 YSLNFMGYV XSXXXXXXVB58 HBV 75 15 POL 580 YSLNFMGYVI XSXXXXXXXI B58 HBV 85 17 POL 746YTSFPWLL XTXXXXXL B58 189 9

TABLE XIV HBV B62 SUPER MOTIFS Source Conservancy Freq Protein PositionSequence String Super Motif Peptide Filed HBV 95 19 POL 521 AICSVVRRAFXIXXXXXXXF B62 HBV 90 18 NUC 58 ALLCWGELM XIXXXXXXM B62 HBV 95 19 POL642 ALMPLYACI XLXXXKXXI B62 3.0012 * HBV 95 19 NUC 54 ALRQAILCWXLXXXXXXW B62 HBV 80 16 ENV 108 AMQWNSTTF XMXXXXXXF B62 HBV 95 19 POL633 APFTQCGY XPXXXXXY B62 19.0013 HBV 95 19 POL 516 AQFTSAICSVXQXXXXXXXV B62 HBV 95 19 POL 516 AQFTSAICSVV XQXXXXXXXXV B62 HBV 100 20ENV 312 CIPIPSSW XIXXXXXW B62 HBV 100 20 ENV 312 CIPIPSSWAF XIXXXXXXXFB62 HBV 90 16 POL 533 CLAFSYMDDV XLXXXXXXXV B62 1.0559 HBV 90 18 POL 533CLAFSYMDDVV XLXXXXXXXXV B62 HBV 85 17 NUC 23 QLGWLWGM XLXXXXXM B62 HBV85 17 NUC 23 CLGWLWGMDI XLXXXXXXXI B62 2.0229 HBV 95 19 ENV 253CLIFLLVLLDY XLXXXXXXXY B62 26.0548 HBV 95 19 ENV 239 CLRRFIIF XLXXXXXFB62 HBV 75 15 ENV 239 CLRRFIIFLF XLXXXXXXXF B62 HBV 75 15 ENV 239CLRRFIIFLFI XLXXXXXXXXI B62 Chisari 4.055 HBV 90 15 NUC 107 CLTFGRETVXLXXXXXXV B62 1.0160 HBV 80 16 X 7 CQLDRADV XQXXXXXXV B62 HBV 85 17 POL622 CQRIVGLLGF XQXXXXXXXF B62 HBV 90 18 NUC 31 DIDPYKEF XIXXXXXF B62 HBV85 17 NUC 29 DLLDTASALY XLXXXXXXXY B62 1.0519 * HBV 95 19 POL 40DLNLGNLNV XLXXXXXXV B62 1.0164 HBV 95 19 POL 40 DLNLGNLNVSI XLXXXXXXXXIB62 HBV 80 16 ENV 122 DPRVRGLY XPXXXXXY B62 HBV 95 19 X 14 DVLCLRPVXVXXXXXV B62 HBV 90 18 POL 541 DVVLGAKSV XVXXXXXXV B62 1.0190 HBV 95 19NUC 43 ELLSFLPSDF XLXXXXXXXF B62 HBV 95 19 NUC 43 ELLSFLPSDFFXLXXXXXXXXF B62 HBV 80 16 ENV 248 FILLLCLI XIXXXXXI B62 Chisari 4.048HBV 80 16 ENV 248 FILLLCLIF XIXXXXXXF B62 HBV 80 16 ENV 246 FIFLLLLCLIXLXXXXXXXI B62 3.0206 HBV 80 16 ENV 246 FLFILLLCLIF XLXXXXXXXXF B62 HBV95 19 POL 513 FLLAQFTSAI XLXXXXXXXI B62 1147.13 * HBV 80 16 ENV 183FLLTRILTI XLXXXXXXI B62 3.0005 * HBV 95 19 ENV 256 FLLVLLDY XLXXXXXY B6226.0027 HBV 95 19 ENV 256 FLLVLLDYQGM XLXXXXXXXXM B62 HBV 75 15 ENV 130FPAGGSSSGTV XPXXXXXXXXV B62 HBV 85 17 ENV 14 FPDHQLDPAF XPXXXXXXXF B6220.0274 HBV 95 19 POL 530 FPHCLAFSY XPXXXXXXY B62 15.0037 * HBV 95 19POL 530 FPHCLAFSYM XPXXXXXXXM B62 15.0217 * HBV 75 15 POL 749FPWLLGCAANW XPXXXXXXXXW B62 HBV 95 19 ENV 346 FVGLSPTV XVXXXXXV B62 HBV95 19 ENV 346 FVGLSPTVW XVXXXXXXW B62 HBV 90 18 X 132 FVLGGCRHKLVXVXXXXXXXXV B62 HBV 95 19 POL 627 GLLGFAAPF XLXXXXXXF B62 20.0124 HBV 9519 POL 509 GLSPFLLAQF XLXXXXXXXF B62 HBV 75 15 ENV 348 GLSPTVWLSVXLXXXXXXXV B62 1.0518 * HBV 75 15 ENV 348 GLSPTVWLSVI XLXXXXXXXXI B62Chisari 4.031 HBV 85 17 NUC 29 GMDIDPYKEF XMXXXXXXXF B62 26.0372 HBV 9519 ENV 173 GPLLVLQAGF XPXXXXXXXF B62 15.0212 HBV 95 19 ENV 173GPLLVLQAGFF XPXXXXXXXXF B62 26.0556 HBV 95 19 NUC 123 GVWIRTPPAYXVXXXXXXXY B62 1.0525 HBV 75 15 POL 569 HLNPNKTKRW XLXXXXXXXW B62 HBV 9018 X 52 HLSLRGLPV XLXXXXXXV B62 1.0212 HBV 80 16 POL 491 HLYSHPIIXLXXXXXI B62 17.0256 HBV 80 16 POL 491 HLYSHPIILGF XLXXXXXXXXF B62 HBV85 17 POL 429 HPAAMPHLLV XPXXXXXXXV B62 20.0273 * HBV 80 16 POL 495HPIILGFRKI XPXXXXXXXI B62 HBV 80 16 POL 497 IILGFRKI XIXXXXXI B6217.0124 * HBV 80 16 POL 497 IILGFRKIPM XIXXXXXXXM B62 HBV 90 18 NUC 59ILCQGELM XLXXXXXM B62 HBV 80 16 POL 498 ILGFRKIPM XLXXXXXXM B62 3.0016HBV 100 20 ENV 249 ILLLCLIF XLXXXXXF B62 HBV 100 20 ENV 249 ILLLCLIFLLVXLXXXXXXXXV B62 Chisari 4.013 HBV 80 16 POL 760 ILRGTSFV XLXXXXXV B62HBV 80 16 POL 760 ILRGTSFVY XLXXXXXXY B62 1.0205 * HBV 80 16 POL 760ILRGTSFVYV XLXXXXXXXV B62 1.0573 * HBV 100 20 NUC 139 ILSTLPETTVXLXXXXXXXV B62 1.0526 * HBV 100 20 NUC 139 ILSTLPETTVV XLXXXXXXXXV B62HBV 90 18 ENV 188 ILTIPQSLDSW XLXXXXXXXXW B62 HBV 100 20 ENV 313IPIPSSWAF XPXXXXXXF B62 15.0032 * HBV 80 16 POL 504 IPMGVGLSPFXPXXXXXXXF B62 HBV 90 18 ENV 191 IPQSLDSW XPXXXXXW B62 19.0004 HBV 90 18ENV 191 IPQSLDSWW XPXXXXXXW B62 15.0030 * HBV 100 20 POL 50 IPWTHKVGNFXPXXXXXXXF B62 15.0209 HBV 90 18 POL 625 IVGLLGFAAPF XVXXXXXKXXF B62 HBV80 16 POL 503 KIPMGVGLSPF XIXXXXXXXXF B62 HBV 85 17 NUC 21 KLCLGWLWXLXXXXXW B62 HBV 85 17 NUC 21 KLCLGWLWGM XLXXXXXXXM B62 3.0209 * HBV 9519 POL 489 KLHLYSHPI XLXXXXXXI B62 3.0009 * HBV 80 16 POL 489 KLHLYSHPIIXLXXXXXXXI B62 HBV 75 15 POL 108 KLIMPARF XLXXXXXF B62 HBV 75 15 POL 108KLIMPARFY XLXXXXXXY B62 1.0171 HBV 80 16 POL 610 KLPVNRPI XLXXXXXI B62HBV 80 16 POL 610 KLPVNRPIDW XLXXXXXXXW B62 HBV 95 19 POL 653 KQAFTFSPTYXQXXXXXXXY B62 20.0256 HBV 95 19 POL 55 KVGNFTGLY XVXXXXXXY B62 1.0166 *HBV 95 19 ENV 254 LIFLLVLLDY XIXXXXXXXY B62 1.0899 HBV 100 20 POL 109LIMPARFY XIXXXXXY B62 26.0028 HBV 95 19 POL 514 LLAQFTSAI XLXXXXXXI B623.0010 * HBV 100 20 ENV 251 LLCLIFLLV XLXXXXXXV B62 1.0835 * HBV 85 17NUC 30 LLDTASALY XLXXXXXXY B62 1.0155 * HBV 90 18 ENV 260 LLDYQGMLPVXLXXXXXXXV B62 1.0516 * HBV 80 16 POL 752 LLGCAANW XLXXXXXW B62 HBV 8016 POL 752 LLGCAANWI XLXXXXXXI B62 3.0013 HBV 95 19 POL 628 LLGFAAPFXLXXXXXF B62 HBV 75 15 ENV 63 LLGWSPQAQGI XLXXXXXXXXI B62 HBV 100 20 ENV250 LLLCLIFLLV XLXXXXXXXV B62 1.0897 * HBV 100 20 ENV 378 LLPIFFCLWXLXXXXXXW B62 HBV 100 20 ENV 378 LLPIFFCLWV XLXXXXXXXV B62 1.0904 * HBV100 20 ENV 378 LLPIFFCLWVY XLXXXXXXXXY B62 26.0549 * HBV 95 19 NUC 44LLSFLPSDF XLXXXXXXF B62 HBV 95 19 NUC 44 LLSFLPSDFF XLXXXXXXXF B62 HBV90 18 POL 407 LLSSNLSW XLXXXXXW B62 HBV 80 16 ENV 184 LLTRILTI XLXXXXXIB62 Chisari 4.053 HBV 95 19 ENV 257 LLVLLDYQGM XLXXXXXXXM B62 3.0207 HBV95 19 ENV 175 LLVLQAGF XLXXXXXF B62 HBV 95 19 ENV 175 LLVLQAGFFXLXXXXXXF B62 20.0121 HBV 100 20 ENV 338 LLVPFVQW XLXXXXXW B62 HBV 10020 ENV 338 LLVPFVQWF XLXXXXXXF B62 HBV 95 19 ENV 338 LLVPFVQWFVXLXXXXXXXV B62 1.0930 * HBV 85 17 NUC 100 LLWFHISCLTF XLXXXXXXXXF B62HBV 95 19 POL 643 LMPLYACI XMXXXXXI B62 17.0130 HBV 100 20 ENV 379LPIFFCLW XPXXXXXW B62 19.0007 * HBV 100 20 ENV 379 LPIFFCLWV XPXXXXXXVB62 15.0034 * HBV 100 20 ENV 379 LPIFFCLWVY XPXXXXXXXY B62 15.0215 HBV100 20 ENV 379 LPIFFCLWVYI XPXXXXXXXXI B62 26.0558 HBV 100 20 POL 123LPLDKGIKPY XPXXXXXXXY B62 15.0210 * HBV 100 20 POL 123 LPLDKGIKPYYXPXXXXXXXXY B62 26.0560 HBV 80 16 POL 611 LPVNRPIDW XPXXXXXXW B62 HBV 8016 POL 611 LPVNRPIDWKV XPXXXXXXXXV B62 HBV 80 16 ENV 178 LQAGFFLLTRIXQXXXXXXXXI B62 HBV 95 19 ENV 258 LVLLDYQGM XVXXXXXXM B62 3.0034 HBV 9519 ENV 176 LVLQAGFF XVXXXXXF B62 HBV 100 20 ENV 339 LVPFVQWF XVXXXXXFB62 HBV 95 19 ENV 339 LVPFVQWFV XVXXXXXXV B62 1.0877 * HBV 90 1 8 NUC119 LVSFGVWI XVXXXXXI B62 Chisari 4.078 HBV 100 20 POL 377 LWDFSQFXVXXXXXF B62 HBV 85 17 ENV 360 MMWYWGPSLY XMXXXXXXXY B62 1039.01 * HBV100 20 POL 1 MPLSYQHF XPXXXXXF B62 19.0010 * HBV 60 16 ENV 109 MQWNSTTFXQXXXXXF B62 HBV 95 19 POL 42 NLGNLNVSI XLXXXXXXI B62 3.0008 HBV 95 19POL 42 NLGNLNVSIPW XLXXXXXXXXW B62 HBV 90 18 POL 406 NLLSSNLSW XLXXXXXXWB62 HBV 95 19 POL 45 NLNVSIPW XLXXXXXW B62 HBV 75 15 ENV 15 NLSVPNPLGFXLXXXXXXXF B62 HBV 90 18 POL 411 NLSWLSLDV XLXXXXXXV B62 1.0185 * HBV 7515 POL 571 NPNKTKRW XPXXXXXW B62 HBV 75 15 POL 571 NPNKTKRWGY XPXXXXXXXYB62 HBV 100 20 POL 47 NVSIPWTHKV XVXXXXXXXV B62 1.0532 HBV 85 17 POL 616PIDWKVCQRI XIXXXXXXXI B62 Chisari 4.091 HBV 85 17 POL 616 PIDWKVCQRIVXIXXXXXXXXV B62 HBV 100 20 ENV 380 PIFFCLWV XIXXXXXV B62 HBV 100 20 ENV380 PIFFCLWVY XIXXXXXXY B62 1.0843 HBV 100 20 ENV 380 PIFFCLWVYIXIXXXXXXXI B62 20.0258 HBV 80 16 POL 496 PIILGFRKI XIXXXXXXI B62 927.48HBV 80 16 POL 496 PIILGFRKIPM XIXXXXXXXXM B62 HBV 100 20 NUC 138PILSTLPETTV XIXXXXXXXXV B62 Chisari 5.125 HBV 100 20 ENV 314 PIPSSWAFXIXXXXXF B62 HBV 100 20 POL 124 PLDKGIKPY XLXXXXXXY B62 1.0174 * HBV 10020 POL 124 PLDKGIKPYY XLXXXXXXXY B62 1.0541 * HBV 100 20 ENV 377PLLPIFFCLW XLXXXXXXXW B62 HBV 100 20 ENV 377 PLLPIFFCLWV XLXXXXXXXXV B62HBV 95 19 ENV 174 PLLVLQAGF XLXXXXXXF B62 HBV 95 19 ENV 174 PLLVLQAGFFXLXXXXXXXF B62 HBV 80 16 POL 505 PMGVGLSPF XMXXXXXXF B62 HBV 95 19 NUC129 PPAYRPPNAPI XPXXXXXXXXI B62 26.0563 HBV 85 17 ENV 58 PPHGGLLGWXPXXXXXXW B62 20.0141 HBV 80 16 ENV 106 PQAMQWNSTT XQXXXXXXXXF B62 HBV90 18 ENV 192 PQSLDSWW XQXXXXXW B62 HBV 85 17 POL 612 PVNRPIDW XVXXXXXWB62 HBV 85 17 POL 612 PVNRPIDWKV XVXXXXXXXV B62 1.0566 HBV 80 16 X 8QLDPARDV XLXXXXXV B62 Chisari 4.116 HBV 95 19 POL 685 QVFADATPTGWXVXXXXXXXXW B62 HBV 90 18 POL 624 RIVGLLGF XIXXXXXF B62 HBV 75 15 POL106 RLKLIMPARF XLXXXXXXXF B62 HBV 75 15 POL 106 RLKLIMPARFY XLXXXXXXXXYB62 HBV 95 19 POL 376 RLWDFSQF XLXXXXXXF B62 20.0122 HBV 80 16 POL 615RPIDWKVCORI XPXXXXXXXXI B62 HBV 90 18 NUC 56 RQAILCWGELM XQXXXXXXXXM B62HBV 90 18 NUC 98 RQLLWFHI XQXXXXXI B62 HBV 75 15 POL 818 RVHFASPLHVXVXXXXXXXV B62 1.0576 HBV 100 20 POL 357 RVTGGVFLV XVXXXXXXV B62 1.0181HBV 100 20 POL 49 SIPWTHKV XIXXXXXV B62 HBV 100 20 POL 49 SIPWTHKVGNFXIXXXXXXXXF B62 HBV 95 19 ENV 194 SLDSWWTSLNF XLXXXXXXXXF B62 HBV 95 19POL 416 SLDVSAAF XLXXXXXF B62 HBV 95 19 POL 416 SLDVSAAFY XLXXXXXXY B621.0186 * HBV 100 20 ENV 337 SLLVPFVQW XLXXXXXXW B62 HBV 100 20 ENV 337SLLVPFVQWF XLXXXXXXXF B62 HBV 95 19 ENV 337 SLLVPFVQWFV XLXXXXXXXXV B62HBV 75 15 POL 581 SLNFMGYV XLXXXXXV B62 HBV 75 15 POL 581 SLNFMGYVIXLXXXXXXI B62 3.0011 HBV 95 19 X 54 SLRGLPVCAF XLXXXXXXXF B62 20.0259HBV 95 19 POL 511 SPFILAQF XPXXXXXF B62 19.0012 * HBV 100 20 NUC 49SPHHTALRQAI XPXXXXXXXXI B62 26.0567 * HBV 75 15 ENV 350 SPTVWLSVXPXXXXXV B62 HBV 75 15 ENV 350 SPTVWLSVI XPXXXXXXI B62 1308.16 HBV 75 15ENV 350 SPTVWLSVIW XPXXXXXXXW B62 1308.17 HBV 75 15 ENV 350 SPTVWLSVIWMXPXXXXXXXXM B62 HBV 75 15 ENV 17 SVPNPLGF XVXXXXXF B62 HBV 80 16 ENV 330SVRFSWLSLLV XVXXXXXXXXV B62 HBV 90 18 POL 739 SWLSRKY XVXXXXXY B6226.0029 HBV 85 17 POL 739 SVVLSRKYTSF XVXXXXXXXXF B62 HBV 90 18 ENV 190TIPQSLDSW XIXXXXXXW B62 HBV 90 18 ENV 190 TIPQSLDSWW XIXXXXXXXW B62 HBV100 20 NUC 142 TLPETTVV XLXXXXXV B62 HBV 100 20 POL 150 TLWKAGILYXLXXXXXXY B62 1.0177 * HBV 90 18 POL 354 TPARVTGGV XPXXXXXXV B6215.0033 * HBV 90 18 POL 35 TPARVTGGVF XPXXXXXXXF B62 15.0214 * HBV 75 15ENV 57 TPPHGGLLGW XPXXXXXXXW B62 1308.04 HBV 75 15 POL 691 TPTGWGLAIXPXXXXXXI B62 HBV 95 19 POL 636 TQCGVPALM XQXXXXXXM B62 HBV 80 16 NUC 16TVQASKLCLGW XVXXXXXXXXW B62 HBV 75 15 ENV 352 TVWLSVIW XVXXXXXW B62 HBV75 15 ENV 352 TVWLSVIWM XVXXXXXXM B62 3.0035 HBV 90 18 X 133 VLGGCRHKLVXLXXXXXXXV B62 1.0589 HBV 95 19 ENV 259 VLLDYQGM XLXXXXXM B62 17.0107HBV 90 18 ENV 259 VLLDYQGMLPV XLXXXXXXXXV B62 1147.14 * HBV 85 17 POL741 VLSRKYTSF XLXXXXXXF B62 HBV 85 17 POL 741 VLSRKYTSFPW XLXXXXXXXXWB62 HBV 95 19 ENV 340 VPFVQWFV XPXXXXXV B62 19.0006 * HBV 80 16 NUC 17VQASKLCLGW XQXXXXXXXW B62 HBV 95 19 ENV 343 VQWFVGLSPTV XQXXXXXXXXV B62HBV 90 18 POL 542 VVLGAKSV XVXXXXXV B62 HBV 86 17 POL 740 VVLSRKYTSFXVXXXXXXXF B62 20.0261 HBV 80 16 POL 759 WILRGTSF XIXXXXXF B62 HBV 80 16POL 759 WILRGTSFV XIXXXXXXV B62 1.0204 * HBV 80 16 POL 759 WILRGTSFVYXIXXXXXXXY B62 1.0572 HBV 80 16 POL 759 WILRGTSFVYV XIXXXXXXXXV B62 HBV95 19 NUC 125 WIRTPPAY XIXXXXXY B62 26.0031 HBV 80 16 POL 751 WLLGCAANWXLXXXXXXW B62 HBV 80 16 POL 751 WLLGCAANWI XLXXXXXXXI B62 Chisari 4.104HBV 95 19 POL 414 WLSLDVSAAF XLXXXXXXXF B62 HBV 95 19 POL 414WLSLDVSAAFY XLXXXXXXXXY B62 26.0551 HBV 100 20 ENV 335 WLSLLVPF XLXXXXXFB62 HBV 100 20 ENV 335 WLSLLVPFV XLXXXXXXV B62 1.0838 * HBV 100 20 ENV335 WLSLLVPFVQW XLXXXXXXXXW B62 HBV 85 17 NUC 26 WLWGMDIDPY XLXXXXXXXYB62 1.0774 * HBV 95 19 ENV 237 WMCLRRFI XMXXXXXI B62 HBV 95 19 ENV 237WMCLRRFII XMXXXXXXI B62 3.0031 * HBV 95 19 ENV 237 WMCLRRFIIF XMXXXXXXXFB62 20.0266 HBV 85 17 ENV 359 WMMWVWGPSL XMXXXXXXXXY B62 26.0552 * HBV100 20 POL 147 YLHTLWKAGI XLXXXXXXXI B62 7.0066 * HBV 100 20 POL 122VLPLDKGI XLXXXXXI B62 HBV 100 20 POL 122 YLPLDKGIKPY XLXXXXXXXXY B6226.0553 HBV 90 18 NUC 118 YLVSFGVW XLXXXXXW B62 HBV 90 16 NUC 118YLVSFGVWI XLXXXXXXI B62 3.0007 * HBV 95 19 POL 640 YPALMPLY XPXXXXXY B6219.0014 * HBV 95 19 POL 640 VPALMPLYACI XPXXXXXXXXI B62 26.0570 242 50

TABLE XV HBV A01 Motif (With binding information) 1st C- ConservancyFrequency Protein Pos Sequence P2 term Peptide Filed A*0101 100 20 POL149 HTLWKAGILY T Y 1069.04 * 0.1100 85 17 NUC 30 LLDTASALY L Y 1069.01 *12.0000 95 19 POL 415 LSLDVSAAFY S Y 1090.07 * 0.0150 75 15 X 103MSTTDLEAY S Y 2.0126 * 0.8500 90 18 POL 738 NSVVLSRKY S Y 2.0123 0.0005100 20 POL 124 PLDKGIKPY L Y 1147.12 * 100 20 POL 124 PLDKGIKPYY L Y1069.03 * 0.1700 85 17 POL 797 PTTGRTSLY T Y 1090.09 * 0.2100 95 19 POL416 SLDVSAAFY L Y 1069.02 * 5.2000 80 16 ENV 119 AMQWNSTTF M F 90 16 POL748 DNSVVLSRKY N Y 20.0255 0.0001 95 19 POL 642 FAAPFTQCGY A Y 20.0254 *0.0680 85 17 POL 590 GYSLNFMGY Y Y 2.0058 95 19 POL 664 KQAFTFSPTY Q Y20.0256 0.0001 85 17 ENV 360 MMWYWGPSLY M Y 1039.01 * 0.0810 100 20 POL165 SASFCGSPY A Y * 16 11

TABLE XVI HBV A03 and A11 Motif (With binding information) Fre- 1stSuper C- Conservancy quency Protein Pos Sequence Motif Motif P2 termPeptide Filed A*0301 A*1101 85 17 POL 721 AACFARSR A03/A11 A03 A R26.0003 0.0004 0.0003 95 19 POL 521 AICSVVRR A03/A11 A03 I R 26.0004−0.0002 0.0003 90 18 POL 772 ALNPADDPSR A03/A11 A03 L R 1.1090 0.00030.0001 85 17 X 70 ALRFTSAR A03/A11 A03 L R 26.0005 0.0047 0.0009 80 16POL 822 ASPLHVAWR A03/A11 A03 S R 75 15 ENV 84 ASTNRCSGR A03/A11 A03 S R1150.60 0.0009 0.0002 80 16 POL 755 CAANWILR A03/A11 A03 A R 85 17 X 69CALRFTSAR A03/A11 A03 A R 26.0149 * 0.0034 0.0230 90 18 X 17 CLRPVGAESRA03/A11 A03 L R 1.1093 0.0011 0.0001 100 20 NUC 48 CSPHHTALR A03/A11 A03S R 5.0055 * 0.0029 0.0001 85 17 NUC 29 DLLDTASALYR A03/A11 A03 L R26.0530 0.0042 −0.0003 85 17 NUC 32 DTASALYR A03/A11 A03 T R 26.00060.0004 −0.0002 95 19 POL 17 EAGPLEEELPR A03/A11 A03 A R 26.0531 −0.0009−0.0003 90 18 POL 718 ELLAACFAR A03/A11 A03 L R 1.0988 0.0002 0.0004 8517 POL 718 ELLAACFARSR A03/A11 A03 L R 26.0532 0.0062 0.0016 95 19 NUC174 ETTVVRRH A03/A11 A03 T R 26.0007 0.0003 −0.0002 80 16 NUC 174ETTVVRRRGR A03/A11 A03 T R 1.1073 0.0003 0.0001 80 16 POL 821 FASPLHVAWRA03/A11 A03 A R 90 18 X 63 FSSAGPCALR A03/A11 A03 S R 95 19 POL 656FTFSPTYK A03/A11 A03 T K 1147.19 * 0.0100 0.0100 95 19 POL 518FTSAICSVVR A03/A11 A03 T R 1.1085 0.0003 0.0003 95 19 POL 518FTSAICSVVRR A03/A11 A03 T R 26.0533 0.0065 0.0092 90 18 X 132 FVLGGCRHKA03/A11 A03 V K 1090.03 * 0.0430 0.0090 75 15 POL 587 GIHLNPNK A03/A11A03 I K 75 15 POL 567 GIHLNPNKTK A03/A11 A03 I K 1.0563 0.0025 0.0011 7615 POL 567 GIHLNPNKTKR A03/A11 A03 I R 85 17 NUC 29 GMDIDPYK A03/A11 A03M K 26.0009 0.0006 0.0004 90 18 POL 735 GTDNSVVLSR A03/A11 A03 T R1090.04 * 0.0010 0.0420 90 18 POL 735 GTDNSVVLSRK A03/A11 A03 T K1147.17 * 0.0140 0.5600 95 19 NUC 123 GVVIRTPPAYR A03/A11 A03 V R26.0535 * 0.1900 0.1700 90 18 NUC 104 HISCLTFGR A03/A11 A03 I R1069.18 * 0.0160 0.0065 75 15 POL 569 HLNPNKTK A03/A11 A03 L K 75 15 POL569 HLNPNKTKR A03/A11 A03 L H 1.0983 0.0025 0.0001 100 20 POL 149HTLWKAGILYK A03/A11 A03 T K 1147.16 * 0.5400 0.4400 90 18 NUC 105ISCLTFGR A03/A11 A03 S R 26.0010 0.0004 0.0002 100 20 POL 153 KAGILYKRA03/A11 A03 A R 26.0011 0.0002 −0.0002 80 16 POL 610 KLPVNRPIDWK A03/A11A03 L K 75 15 X 130 KVFVLGGCR A03/A11 A03 V R 1.0993 * 0.0420 0.0820 8517 POL 720 LAACFARSR A03/A11 A03 A R 20.0129 0.0058 0.0065 90 18 POL 719LLAACFAR A03/A11 A03 L R 26.0012 0.0024 0.0003 85 17 POL 719 LLAACFARSRA03/A11 A03 L R 85 17 NUC 30 LLDTASALYR A03/A11 A03 L R 1.1070 0.00500.0002 80 16 POL 752 LLGCAANWILR A03/A11 A03 L R 75 15 POL 564LSLGIHLNPNK A03/A11 A03 S K 95 19 NUC 169 LSTLPETTVVR A03/A11 A03 S R26.0537 −0.0009 0.0008 75 15 POL 3 LSYCHFRK A03/A11 A03 S K 85 17 POL 99LTVNEKRR A03/A11 A03 T R 26.0013 −0.0002 −0.0002 90 18 NUC 119 LVSFGVWIRA03/A11 A03 V R 1090.08 * 0.0028 0.0120 100 20 POL 377 LWDFSQFSR A03/A11A03 V R 1069.20 * 0.0016 0.3600 75 15 X 103 MSTTDLEAYFK A03/A11 A03 S K90 18 NUC 75 NLEDPASR A03/A11 A03 L R 26.0014 −0.0002 −0.0002 95 19 POL45 NLNVSIPWTHK A03/A11 A03 L K 26.0538 −0.0009 0.0005 90 18 POL 738NSVVLSRK A03/A11 A03 S K 26.0015 0.0006 0.0010 100 20 POL 47 NVSIPWTHKA03/A11 A03 V K 1069.16 * 0.0820 0.0570 90 18 POL 775 PADDPSAGR A03/A11A03 A R 1150.35 0.0008 0.0002 80 16 X 11 PARDVLCLR A03/A11 A03 A R1150.36 0.0002 0.0002 75 15 EVN 83 PASTNRQSGR A03/A11 A03 A R 90 18 POL618 PIDWKVCWQR A03/A11 A03 I R 1.0985 0.0002 0.0005 80 16 POL 498PIILGFRK A03/A11 A03 I K 95 19 POL 20 PLEEELPR A03/A11 A03 L R 26.00160.0002 −0.0002 100 20 POL 2 PLSYQHFR A03/A11 A03 L R 26.0017 −0.0002−0.0002 75 15 POL 2 PLSYQHFRK A03/A11 A03 L K 1.0161 0.0011 0.0031 85 17POL 98 PLTVNEKR A03/A11 A03 L R 26.0018 0.0002 −0.0002 85 17 POL 98PLTVNEKRR A03/A11 A03 L R 1.0974 0.0008 0.0005 90 18 X 20 PVGAESRGRA03/A11 A03 V R 1.0990 0.0002 0.0005 85 17 POL 612 PVNRPIDWK A03/A11 A03V K 1142.06 * 0.0310 0.1400 95 19 POL 654 QAFTFSPTYK A03/A11 A03 A K1090.10 * 0.0450 0.5400 80 16 EVN 179 QAGFFLLTR A03/A11 A03 A R 75 15NUC 169 QSPRFFFSQSR A03/A11 A03 S R 28.0839 80 16 POL 189 QSSGILSRA03/A11 A03 S R 75 15 POL 106 RLKLIMPAR A03/A11 A03 L R 1.0975 * 0.09500.0002 75 15 X 128 FLKVFVLGGCR A03/A11 A03 L R 95 19 POL 376 FLWDPSQFSRA03/A11 A03 L R 26.0539 * 0.2800 3.8000 95 19 NUC 183 RSPRRRTPSPRA03/A11 A03 S A 26.0540 −0.0007 −0.0003 75 15 NUC 167 RSQSPARR A03/A11A03 S R 75 15 NUC 187 RSQSPARRR A03/A11 A03 S R 95 19 NUC 188 RTPSPARRA03/A11 A03 T R 26.0019 −0.0002 −0.0002 95 19 NUC 188 RTPSPRARR A03/A11A03 T R 1.0971 * 0.0054 0.0005 100 20 POL 357 RVTGGVFLVDK A03/A11 A03 VK 1147.18 * 0.0190 0.0290 90 18 X 65 SAGPCALA A03/A11 A03 A R 28.0020−0.0002 0.0020 95 19 POL 520 SAICSVVR A03/A11 A03 A R 26.0021 −0.00020.0071 95 19 POL 520 SAICSVVRR A03/A11 A03 A R 1090.11 * 0.0058 0.210090 18 POL 771 SALNPADDPSR A03/A11 A03 A R 26.0542 −0.0004 −0.0003 75 15POL 565 SLGHLNPNK A03/A11 A03 L K 28.0758 * 90 18 X 64 SSAGPCALA A03/A11A03 S R 26.0153 * 0.0080 0.1400 95 19 NUC 170 STLPETTVVR A03/A11 A03 T R1069.21 * 0.0007 0.0600 95 19 NUC 170 STLPETTVVRR A03/A11 A03 T R1083.01 0.0150 1.4000 80 16 ENV 85 STINRQSGR A03/A11 A03 T R 75 15 X 104STTDLEAYFK A03/A11 A03 T K 1.0584 * 0.0066 2.7000 85 17 POL 716TAELLAACFAR A03/A11 A03 A R 26.0544 0.0006 0.0023 95 19 NUC 171TLPETTVVR A03/A11 A03 L R 1.0969 0.0008 0.0002 95 19 NUC 171 TLPETTVVRRA03/A11 A03 L R 1069.22 * 0.0007 0.0230 95 19 NUC 171 TLPETTVVRRRA031A11 A03 L R 26.0545 * 0.0005 0.0160 100 20 POL 150 TLWKAGILYKA03/A11 A03 L K 1069.15 * 5.3000 0.3600 100 20 POL 150 TLWKAGILYKRA03/A11 A03 L R 26.0546 0.0082 0.0095 95 19 POL 519 TSAICSVVR A03/A11A03 S R 5.0057 0.0005 0.0008 95 19 POL 519 TSAICSVVAR A03/A11 A03 S R1142.08 * 0.0018 0.0006 75 15 X 105 TTDLEAYFK A03/A11 A03 T K 1.0215 *0.0006 0.9200 75 15 EVN 278 TTSTGPCX A03/A11 A03 T K 80 16 NUC 175TTVVRRRGR A03/A11 A03 T R 1.0970 0.0008 0.0005 80 16 NUC 176 TVVRRRGRA03/A11 A03 V R 3.0324 0.0003 0.0001 80 16 NUC 176 TVVRRRGRSPR A03/A11A03 V R 28.0837 90 18 X 133 VGGCRHK A03/A11 A03 L K 26.0022 0.01500.0002 80 16 EVN 177 VLQAGFFLLTR A03/A11 A03 L R 90 18 NUC 120 VSFGWWRA03/A11 A03 S R 26.0023 * 0.0040 0.0290 100 20 POL 48 VSIPWTHK A03/A11A03 S K 26.0024 * 0.0130 0.0170 100 20 POL 358 VTGGVFLVDK A03/A11 A03 TK 1069.17 * 0.0390 0.0920 100 20 POL 378 WDFSQFSR A03/A11 A03 V R1069.19 * 0.0015 0.0750 80 16 NUC 177 WRRRGRSPR A03/A11 A03 V R 1.10740.0027 0.0001 80 16 NUC 177 WRRRGRSPRR A03/A11 A03 V R 28.0838 95 19 NUC125 WIRTPPAYR A03/A11 A03 I R 1.0968 0.0008 0.0005 90 18 POL 314WLQFRNSK A03/A11 A03 L K 26.0025 −0.0002 0.0005 85 17 NUC 26 WLWGMDIDPYKA03/A11 A03 L K 26.0547 0.0030 0.0013 100 20 POL 122 YLPLDKGIK A03/A11A03 L K 1.0173 0.0001 0.0001 90 18 NUC 118 YLVSFGVWIR A03/A11 A03 L R1090.13 * 0.0005 0.0002 90 18 POL 538 YMDDVVLGAK A03/A11 A03 M K1090.15 * 0.0330 0.0043 80 16 POL 493 YSHPILGFR A03/A11 A03 S R 80 16POL 493 YSHPIILGFRK A03/A11 A03 S K 95 19 POL 643 AAPFTQCGY A03/A11 A03A Y 95 19 POL 540 AFPHCLAFSY A03/A11 A03 F Y 95 19 X 62 AFSSAGPCAA03/A11 A03 F A 95 19 POL 866 AFTFSPTYK A03/A11 A03 F K 20.0130 * 0.26000.0400 95 19 POL 666 AFTFSPTYKA A03/A11 A03 F A 95 19 POL 18 AGFLEEFLPRA03/A11 A03 G R 20.0265 0.0004 0.0002 95 19 POL 532 AICSVVRRAF A03/A11A03 I F 80 16 ENV 119 AMQWNSTTF A03/A11 A03 M F 80 16 ENV 119 AMQWNSTTFA03/A011/A24 A03 M F 80 16 ENV 119 AMQWNSITRH A03/A11 A03 M H 85 17 POL734 CFARSRSGA A03/A11 A03 F A 75 15 POL 618 CFRKLPVNR A03/A11 A03 F R 9519 POL 649 CGYPALMPLY A03/A11 A03 G Y 100 20 EVN 323 CIPIPSSVVAF A03/A11A03 I F 75 15 ENV 239 CLRRHFIFLF A03/A11 A03 L F 95 19 POL 534CSVVRRAFPH A03/A11 A03 S H 85 17 NUC 58 DLLDTASALY A03/A11 A03 L Y1.0519 * 0.0001 0.0001 95 19 ENV 207 DSVVWTSLNF A03/A11 A03 S F 20.01200.0006 0.0002 95 19 NUC 43 ELLSFLPYSDF A03/A11 A03 L F 95 19 NUC 72ESPEHCSPH A03/A11 A03 S H 95 19 NUC 72 ESPEHCSPHH A03/A11 A03 S H 95 19POL 642 FAAPFTQCGY A01/A03/A11 A03 A Y 20.0254 * 90 18 ENV 24 FFPDHCLDPAA03/A11 A03 F A 75 15 NUC 139 FGRETVLEY A03/A11 A03 G Y 75 15 POL 255FGVEFSGSGH A03/A11 A03 G H 80 16 ENV 248 FILLLCUF A03/A11 A03 I F 100 20ENV 344 FSVVLSLLVPF A03/A11 A03 S F 20.0263 0.0004 0.0002 95 19 POL 867FTFSPTYKAF A03/A11 A03 T F 20.0262 0.0004 0.0006 80 16 POL 765 GCAANWILRA03/A11 A03 C R 95 19 POL 638 GLLGFAAPF A03/A11 A03 L F 20.0124 0.00060.0002 95 19 POL 520 GLSPFLLAQF A03/A11 A03 L F 85 17 NUC 29 GMDIDPYKEFA03/A24 A03 M F 26.0372 −0.0003 −0.0002 80 16 POL 258 GVEPSGSGH A03/A11A03 V H 100 20 POL 372 GVFLVDKNPH A03/A11 A03 V H 95 19 NUC 152GVWIRTPPAY A03/A11 A03 V Y 1.0525 0.0047 0.0002 100 20 NUC 78 HCSPHHTALRA03/A11 A03 C R 80 18 POL 831 HFASPLHVA A03/A11 A03 F A 85 17 POL 728HTAELLAACF A03/A11 A03 T F 95 19 POL 533 ICSVVRRAF A03/A11 A03 C F 95 19ENV 266 IFLLVLLDY A03/A11 A03 F Y 80 16 POL 771 ILRGTSFVY A03/A11 A03 LY 1.0205 * 0.0440 00002 75 15 POL 108 KLIMPARFY A03/A11 A03 L Y 1.017175 15 X 130 KVFVGGCRH A03/A11 A03 V H 95 19 POL 55 KVGNFTGLY A03/A11 A03V Y 1142.05 * 0.2100 0.0170 100 20 POL 125 LDKGIKPYY A03/A11 A03 D Y 9519 ENV 206 LDSWWTSLNF A03/A11 A03 D F 85 17 NUC 60 LDTASALYR A03/A11 A03D R 26.0151 0.0004 −0.0002 95 19 POL 428 LDVSAAFYH A03/A11 A03 D H 80 16EVN 247 LFILLLCLIF A03/A11 A03 F F 80 16 ENV 247 LFILLLCLIF A03/A24 A03F F 80 16 POL 764 LGCAANVVLR A03/A11 A03 G R 75 15 POL 577 LGHLNPNKA03/A11 A03 G K 95 19 ENV 265 LIFLLVLLDY A03/A11 A03 I Y 1.0899 0.00220.0004 95 19 NUC 44 LLSFLPSDF A03/A11 A03 L F 95 19 NUC 44 LLSFLPSDFFA03/A11 A03 L F 95 19 ENV 175 LLVLQAGFF A03/A11 A03 L F 20.0121 0.00060.0002 100 20 ENV 349 LLVPFVQWF A03/A11 A03 L F 95 19 NUC 45 LSFLPSDFFA03/A11 A03 S F 20.0123 0.0006 0.0002 95 19 POL 426 LSLDVSAAF A03/A11A03 S F 95 19 X 53 LSLRGLPVCA A03/A11 A03 S A 95 19 POL 521 LSPFLLAQFA03/A11 A03 S F 75 15 ENV 18 LSVPNPLGF A03/A11 A03 S F 100 20 POL 423LSWLSLDVSA A03/A11 A03 S A 20.0260 0.0048 0.0035 95 19 ENV 249 MCLRRFIIFA03/A11 A03 C F 90 18 POL 550 MDDVVLGAK A03/A11 A03 D K 90 18 NUC 30MDIDIPYKEF A03/A11 A03 D F 85 17 ENV 360 MMWYWGPSLY A01/A03/A11 A03 M Y1039.01 * 0.0500 0.0008 75 15 X 103 MSTTDLEAYF A03/A11 A03 S F 95 19 POL572 NFLLSLGIH A03/A11 A03 F H 95 19 POL 45 NLNVSIPWTH A03/A11 A03 L H 7515 ENV 15 NLSVPNPLGF A03/A11 A03 L F 75 15 ENV 215 NSQSPTSNH A03/A11 A03S H 90 18 POL 385 PARVTGGVF A03/A11 A03 A F 85 17 X 68 PCALRFTSARA03/A11 A03 C R 90 18 ENV 26 PDHQLDPAF A03/A11 A03 D F 95 19 POL 523PFLLAQFTSA A03/A11 A03 F A 95 19 POL 645 PFTQCGYPA A03/A11 A03 F A 10020 ENV 244 PGYRWMCLR A03/A11 A03 G A 1.0964 0.0008 0.0005 95 19 ENV 244PGYRWMCLRR A03/A11 A03 G R 1.1068 0.0048 0.0001 100 20 ENV 391 PIFFCLWVYA03/A11 A03 I Y 1.0843 0.0011 0.0002 100 20 POL 438 PLHPAAMPH A03/A11A03 L H 20.0128 0.0012 0.0002 95 19 ENV 174 PLLYLQAGF A03/A11 A03 L F 9519 ENV 174 PLLVQAGFF A03/A11 A03 L F 80 16 POL 516 PMGVGLSPF A03/A11 A03M F 80 16 POL 516 PMGVGLSPF A03/A24 A03 M F 95 19 POL 665 QAFTFSPTYA03/A11 A03 A V 20.0127 0.0030 0.0017 80 18 ENV 118 QAMQWNSTTF A03/A11A03 A F 95 19 POL 539 RAFPHCLAF A03/A11 A03 A F 20.0125 0.0015 0.0007 7515 POL 106 RLKLIMPARF A03/A11 A03 L F 95 19 POL 387 PLVVDFSCF A03/A11A03 L F 20.0122 0.0006 0.0002 80 18 POL 829 RVHFASPLH A03/A11 A03 V H 9018 X 65 SAGPCALRF A03/A11 A03 A F 26.0152 −0.0003 0.0004 100 20 POL 165SASFCGSPY A01/A03/A11 A03 A Y * 75 15 POL 759 SFPWLLGCA A03/A11 A03 F A75 15 POL 769 SFPWLLGCAA A03/A11 A03 F A 95 19 POL 427 SLDVSAAFYHA03/A11 A03 L H 100 20 ENV 348 SLLVPFVQWF A03/A11 A03 L F 95 19 X 54SLRGLPVCAF A03/A11 A03 L F 20.0259 0.0004 0.0002 90 18 X 64 SSAGPCALRFA03/A11 A03 S F 26.0374 −0.0003 −0.0002 75 15 X 104 STTDLEAYF A03/A11A03 T F 95 19 POL 535 SVVRRAFPH A03/A11 A03 V H 20.0131 * 0.1100 0.610085 17 POL 727 TAELLAACF A03/A11 A03 A F 90 18 POL 747 TDNSVVLSR A03/A11A03 D R 90 18 POL 747 TDNSVVLSRK A03/A11 A03 D K 20.0264 0.0006 0.001775 15 NUC 138 TFGRETVLEY A03/A11 A03 F Y 95 19 POL 688 TFSPTYKAF A03/A24A03 F F 5.0064 100 20 POL 370 TGGVFLVDK A03/A11 A03 G K 20.0133 0.00070.0061 100 20 POL 150 TLWKAGILY A03/A11 A03 L Y 1099.03 * 0.1300 0.000875 15 POL 756 TSFPWLLGCA A03/A11 A03 S A 80 16 POL 775 TSFVYVPSA A03/A11A03 S A 100 20 POL 373 VFLVDKNPH A03/A11 A03 F H 80 18 X 131 VFVLGGCRHA03/A11 A03 F H 75 15 X 131 VFVLGGCRHK A03/A11 A03 F K 95 19 POL 637VGLLGFAAPF A03/A11 A03 G F 85 17 POL 96 VGPLTVNEK A03/A11 A03 G K20.0132 0.0007 0.0078 85 17 POL 98 VGPLTVNEKR A03/A11 A03 G R 95 19 POL554 VLGAKSVQHI A03/A11 A03 L H 85 17 POL 752 VLSRKYTSF A03/A11 A03 L F90 18 POL 553 VVLGAKSVQH A03/A11 A03 V H 85 17 POL 751 VVLSRKYTSFA03/A11 A03 V F 20.0261 0.0004 0.0002 90 18 NUC 131 WFHISCLTF A03/A11A03 F F 13.0073 * 90 18 NUC 131 WFHISCLTF A03/A24 A03 F F 13.0073 * 8517 NUC 28 WGMDIDPYK A03/A11 A03 G K 26.0154 −0.0003 0.0006 85 17 POL 589WGYSLNFMGY A03/A11 A03 G Y 80 16 POL 770 WILRGTSFVY A03/A11 A03 I Y1.0572 0.0076 0.0011 95 19 POL 425 WLSLDVSAAF A03/A11 A03 L F 85 17 NUC26 WLWGMDIDPY A03/A11 A03 L Y 1.0774 * 0.0002 0.0002 95 19 ENV 248WMCLRRFIIF A03/A11 A03 M F 20.0266 0.0004 0.0011 95 19 ENV 248WMCLRRRIIF A03/A24 A03 M F 20.0266 0.0004 0.0011 80 16 POL 504 YSHPILGFA03/A11 A03 S F 248 47

TABLE XVII HBV A24 Motif (With binding information) 1st C- ConservancyFrequency Protein Pos Sequence P2 term Peptide Filed A*2402 100 20 NUC131 AYRPPNAPIL Y L 1069.24 * 0.0042 95 19 ENV 234 GYRWMCLRRF Y F1069.25 * 85 17 POL 745 KYTSFPWLL Y L 1069.23 * 5.3000 80 16 POL 492LYSHPIILGF Y F 2.0181 * 1.1000 75 15 POL 4 SYQHFFKLL Y L 2.0042 75 15POL 4 SYQHFRKLLL Y L 2.0173 * 0.0660 95 19 X 62 AFSSAGPCAL F L 5.0118 9018 POL 546 AFSYMDDVVL F L 13.0130 80 18 ENV 119 AMQWNSTTF M F 100 20 NUC160 AYRPPNAPI Y I 1090.02 * 0.0310 90 18 NUC 146 EYLVSFGVW Y W 26.015090 18 NUC 146 EYLVSFGVWI Y I 17.0426 * 80 16 ENV 182 FFLLTRILTI F I 8018 ENV 181 GFFLLTRIL F L 75 15 ENV 170 GFLGPLLVL F L 85 17 NUC 29GMDIDPYKEF M F 25.0372 85 17 ENV 65 GWSPQAQGI W I 20.0134 85 17 ENV 65GWSPQAQGIL W L 20.0268 80 16 POL 831 HFASPLHVAW F W 100 20 ENV 392IFFCLWVYI F I 5.0058 80 16 ENV 245 IFLFILLLCL F L 95 19 POL 406KFAVPNLQSL F L 5.0114 100 20 POL 121 KYLPLDKGI Y I 80 16 ENV 247LFILLLCLI F I 80 16 ENV 247 LFILLLCLIF F F 85 17 NUC 130 LWFHISCLTF W F26.0373 95 19 POL 572 NFLLSLGIHL F L 5.0115 80 16 POL 769 NWILRGTSF W F95 19 POL 645 PFTQCGYPAL F L 5.0116 95 19 ENV 352 PFVQWFVGL F L 5.005980 16 POL 516 PMGVGLSPF M F 80 16 POL 761 PWLLGCAANW W W 100 20 POL 51PWTHKVGNF W F 20.0138 * 0.0290 75 15 ENV 242 RFIIFLFIL F L 75 15 ENV 242RFIIFLFILL F L 95 19 ENV 247 RWMCLRRFI W I 20.0135 * 0.0710 95 19 ENV247 RWMCLRRFII W I 20.0269 * 1.1000 100 20 POL 167 SFCGSPYESW F W20.0139 * 0.0710 80 16 POL 776 SFVYVPSAL F L 100 20 ENV 345 SWLSLLVPF WF 20.0136 * 0.3900 95 19 POL 403 SWPKFAVPNL W L 20.0271 * 5.6000 95 19ENV 208 SWWTSLNFL W L 20.0137 * 0.3800 95 19 POL 668 TFSPTYKAF F F5.0064 95 19 POL 668 TFSPTYKAFL F L 5.0117 95 19 POL 697 VFADATPTGW F W20.0272 * 0.0180 90 18 NUC 131 WFHISCLTF F F 13.0073 * 0.0300 95 19 ENV356 WFVGLSPTVW F W 20.0270 * 0.0120 95 19 ENV 248 WMCLRRFIIF M F 20.026648 16

TABLE XVIII DR SUPER MOTIF (With Binding information) Sequence PeptideDR1 DR2w2B1 IDR2w2b2 DR3 DR4w4 DR4w15 DR5w11 DR5w12 DR6w19 DR7 DR8w2 DR9DRw53 LLGFAAPFTQCGYPA CQVFADATGWGLA WPKFAVPNLQSLTNL 1188.30 0.00070.0013 0.0023 0.0002 0.0008 0.0180 CLTFGRETVLETLVS RRSFGVEPSGSGHDLLWFHISCLTFGRET MOLFHLCUISCSCP IFLFLLLCLIFLLV 1280.11 0.0005 0.00410.0018 FIIRFILLLCLIFL TSGFGPLLYLQAGF AGFFLLTRILTIPQS 1280.06 4.60000.0420 0.0190 0.0040 5.3000 0.1500 3.6000 0.0700 0.3700 3.1000 0.26001.3000 CLIFLLVLLDYQGML GLYFPAGGSSSSTVN LGFFPDHQLDPAFGA RRAFPHLAFSYMDDF107.05 0.0010 0.0010 −0.0009 0.0010 0.0017 LGFFKIFMGVGLSPKQQFRKLPVINFPIDW 1298.04 1.5000 0.0022 0.0210 −0.0006 1.2000 0.85000.0130 0.0013 0.0043 0.4000 0.0580 0.0250 VCAFSSAGPCALFFT 1186.29 0.21000.2600 0.0023 0.0003 0.0200 0.0150 SVRFSWLSLLVPFVQ 1280.20 0.9000 0.00990.0037 KQAFTFSPTYKAFLC 1298.08 0.5300 0.2400 0.1400 0.0090 1.1000 0.22000.2400 0.0024 0.0200 0.3300 0.1200 0.5400 VGNFTGLYSSTVPVF 1298.02 1.70000.0100 0.0016 0.0140 0.1700 0.0035 0.0580 0.5600 0.0044 0.3100LAQFTSAICSVVRRA 1186.10 0.0120 0.0085 0.1500 −0.0009 0.0150 0.02800.0076 0.0091 0.0010 0.0280 0.0150 0.0880 0.0190 VQWFVGLSPTVWLSVLKVFVLGGCRHKLVC LVPFVQWFVGLSPTV 1186.15 0.0130 0.6900 0.0140 −0.00130.1500 1.4000 0.3800 0.6600 0.0018 0.0092 0.6800 2.5000 2.6000GTSPVYVPSALNPAD 1280.09 0.3500 0.0140 0.0500 −0.0006 0.3800 0.41000.0470 −0.0001 0.0001 0.2700 0.0610 0.3400 NRPDWKVCQRIVGL FFIFLLLCLIFLCUFLLVLLDYQGM F107.02 0.0016 0.0060 0.0230 0.0017 0.0044AKLIGTDNSVVLSRK PLPIHTAELLAACFA 1280.181 0.0046 0.0490 −0.0003RRFIIFLLLCU FLFILLLCLIFLLVL AWWILRGTSFVYVPS NAPILSTLPETTVVR 1186.160.0009 0.0009 −0.0007 −0.0002 0.0005 0.1600 CTCIPPSSWAFARFGVVIRTPPYRPPNA 27.0280 0.3700 0.0420 7.2000 0.0120 3.4000 0.5700 0.48000.0140 −0.0004 0.2200 0.5300 0.0450 AELLAACFARSRSGA PHCLAFSYMDDVVLGPFLLAQFTSAVCSVV F107.04 0.1800 0.0270 0.0042 −0.0013 0.0800 0.12000.0120 0.0016 0.0800 0.0770 0.0580 0.0590 ASKLCLGWLWGMDID 1186.03 0.0002−0.0005 0.0017 −0.0002 0.0013 0.0010 ILLLCLIFLLVLLDY F107.01 0.00280.0069 0.0320 0.0018 0.0047 FDVLCLRPVGAESRG FPGLCQVFADATPTGPQSLDSWWTSLNFLG RDLLDTASALYREAL 1280.19 0.0001 0.0092 0.0770WLSLDVSAAFYHIPL LVLLDYQGMLPVCPL 1280.17 0.0034 −0.0013 0.0011AGPLEEELPRLADEG 35.0091 0.0022 ILLFLFILLLCLIFLL 1280.12 DVVLGAKSVQHLESLVGLLGFAAPFTQCGY 1280.21 0.0470 0.3100 0.0008 −0.0014 −0.0004 −0.00010.0014 0.5700 PILLGFRKIPMGVGL DLNLGNLNVSIPWTH 1280.07 0.0038 0.02400.0010 SGFLGPLLVLQAGFF HLPLHPAAMPHLLVG LLCLIFLLVLLDYQG 1280.16KRRLIKLIMPARFYPN EIFLKVFVLGGCRHK SPFLLAQFTSAICSV 1186.26 0.1200 0.02000.0085 −0.0013 0.0740 0.0190 −0.0002 −0.0013 0.0540 0.0330 0.0014 0.03800.2000 IRDLLDTASALYREA FPWLLGCAANWILRG IVGLLGFAAPFTQCG 1188.09 0.0200−0.0005 −0.0007 −0.0002 0.0009 0.0067 HGGLLGWSPQACGIL LFILLLCLIFLLVLLSVELLSPFLPSDFFPS TNFLLSLGIHLINPK 1298.03 3.5000 0.0410 0.1200 0.02200.0360 0.0053 0.0160 0.2200 0.0032 0.3800 LTNLLSSNLSWLSLD 1186.14 0.00100.0083 0.0160 0.0013 0.0019 0.0200 GFFLLTRLTIPQSL 1280.08 0.4300 0.01500.0110 3.1000 0.4500 2.3000 0.0780 3.5000 1.8000 0.5500 LGPLLVLQAGFFLLTWLSLLVPFVQWFVGL IRQLLWFHSCLTFG YPALMPLYACIQSKQ 1298.05 0.2400 0.00140.0011 AEDLNLGNLVVSIPW 1166.01 0.0001 −0.0005 −0.0007 −0.0002 −0.00030.0170 GHLNPNKTKRWGYS DEGLNRRVAEDLNLG LGNLVVSIPWTHKVG LSTLPETVVRRRGRLPLLPIFFCLWVYIZ VAPLPIHTAELLAAC FFKLPVNRPIDWKYC SWWLQFRNSKPCSDYHLSLRLPCAFSSA 1280.10 1.3000 0.0028 0.0130 VLCLRPVGAERGRPHTALRQAILCWGELM WMCLIRRRFIRLFILL VELLSFLPSDFFPSI LSWLSLDVSAAFYHIFSWLSLLVPFVQWFV GAHLSLRGLIPVCAFS 1186.07 0.7800 0.0042 −0.0041 0.00110.0025 0.0077 0.0150 GVGLSPFLLAQFTSA SVVLSRKYTSFPWLL 27.0282 0.00050.0057 0.2100 −0.0016 0.5300 0.0130 TNLLSSNLSWLSLDV 1186.26 0.0016−0.0005 0.1300 0.0008 0.0019 0.0410 GTNLSVPNPLGFFPD SSNLSWLSLDVSAAF1186.27 0.1400 0.0030 −0.0005 1.5000 0.2700 0.0046 0.0180 0.1000 0.00390.0460 0.0110 6.2000 TRILTIPQSLDSWWT LQSLTNLLSSNLSWL F107.03 2.50000.4400 0.0200 −0.0013 4.8000 0.8100 0.0680 0.7500 0.0260 0.1500 0.08800.1100 FFLLTRILTIPQSLD F064.01 GVFLVDKNPHNTTES LEYLVSFGWWIRTPPWSRLVDFSQFSPGN 35.0098 2.6000 RQLLWFHSCLTFGR 1186.23 0.0002 0.00090.0140 0.0011 0.0081 0.0096 LGWLWGMDIDPYKEF 1186.12 0.0004 0.0006 0.02000.0280 −0.0002 0.0004 0.0430 LHTLWKAGILYKRET ASALYREALESPEHCKLHLYSHPILGFRK FSYMIDDVVLGAKSVQ KIPMGVGLSPFLLAQ PAAMPHLLVGSSSGLSPQAMOMNSTTFHQTL 1298.01 0.0012 0.0300 0.1200 LSAMSTTDLEAYFKDIWMMWYWGPSLYNIL GLPVCAFSSAGPCAL DVWKVCQRIVGLLSPA 1186.05 0.0120 −0.00260.0030 0.2500 0.0018 0.0130 LCQVFADATPTGWGL 1280.14 0.0020 0.9600 0.0013QWFVGLSPTWLSVI QQYVGPLTVNEKRRL PDRVHFASPLHVAWR 1298.08 0.0510 0.02900.0008 0.0008 0.0054 0.0008 0.0190 0.0810 0.0035 0.2400 ARDVLCLRPVGAESRDDVVLGAKSVQHLES LPKVLHKRTLGLSAM KFAVPNLQSLTMLLS 1280.13 0.0180 0.0005−0.0003 0.1300 0.0043 0.0088 −0.0003 0.0056 CPTVQASKLCLGWLWWASVRFSWLSLLYPF CSVVRRAFPHCLAFS 1186.04 0.1000 0.1024 0.0770 0.00320.0016 −0.0022 0.0008 −0.0013 0.0540 0.0590 0.0250 1.2000 0.0460NLWSIPWTHKVGNF 1186.17 0.0001 −0.0005 −0.0041 −0.0007 −0.0002 0.00050.0009 SFGVVIRTPPAYRPP 1186.25 0.0094 0.0110 0.4300 −0.0009 0.07800.0630 0.0260 0.0071 0.0002 0.0240 0.2500 0.0800 0.0018 TSFVYVPSALNPADDCLLWRHSCLTFGRE FVQWFVGLSPTVWLS 1186.08 0.4700 0.0035 0.0180 −0.00130.0130 0.0072 0.0021 0.0190 0.0690 0.0180 0.0410 0.0044 AAWWWLRGTSFVYVP1298.07 0.0920 0.0240 0.0061 0.0023 0.0510 0.0250 0.0140 0.3700 0.02500.5800 0.2500 0.2700 FGVWRITPAYRPPN HTLWKAGLYKRETT SFPWLLGCAANWILRNLSWLSLDSAAFYH 1186.18 0.1400 0.0003 −0.0005 1.3000 0.2900 0.0033 0.00220.0330 0.0041 0.0150 0.0620 2.4000 RFSWLSLLVPFVQWF 1186.22 0.0430 0.0009−0.0007 0.0002 0.0005 0.0031 RVSWPKFAVPNLQLS AFSYMDDVVLGAKSV 1186.020.0027 −0.0005 0.0130 2.9000 0.0008 −0.0003 −0.0005 QCGYPALMPLYACIQ1186.21 0.0062 0.0018 0.0068 0.0023 0.0006 LLDYQGMLPVCPLIPPPAYRPPNAPILSTL 1186.20 0.0056 −0.0005 0.0038 0.0022 0.0024 0.0015CPGYRMMCLIFFFF LHLYSHPILLGFRKI 1280.15 0.0220 0.0340 0.0400 0.00400.6800 0.1600 0.0410 0.0310 0.0002 0.0006 0.0610 0.0490 RQGYSLINFMGYVIGSSFVYVPSALNPADDP 145

TABLE XIX Total Core Conservancy Total Freq. Conversancy Core FreqProtein Position Core Sequence 90.00 18 90.00 18 X 46 AHLSLRGLP 5.00 175.00 1 ENV 172 AVLDPRVRG 45.00 9 95.00 19 ENV 10 FFPDHCLDP 70.00 1475.00 15 NUC 136 FGRETVLEY 30.00 6 75.00 15 POL 241 FGVEPSGSG 55.00 11100.00 20 POL 360 FLVDKNPHN 55.00 11 95.00 19 POL 655 FSPTYKAFL 65.00 1380.00 16 POL 731 IGTDNSVVL 100.00 20 100.00 20 POL 47 IPWTHKVGN 65.00 1390.00 18 POL 18 LEEELPFLA 100.00 20 100.00 20 POL 120 LPLDKGIKP 55.00 1195.00 19 POL 412 LSLDVSAAF 60.00 12 65.00 17 POL 98 LTVNEKRRL 45.00 9100.00 20 POL 374 LVVDFSDFQ 85.00 17 85.00 17 NUC 34 LYREALESP 45.00 985.00 17 NUC 27 MDIDPYKEF 85.00 17 100.00 20 POL 34 VAEDLNLGN 95.00 1995.00 19 POL 683 VFADATPTG 35.00 7 95.00 19 X 13 VGAESRGRP 90.00 1895.00 19 ENV 256 VLLDYQGWL 85.00 17 90.00 18 POL 737 WLSRKYTS 90.0 1890.00 16 POL 535 TMDDVVLGA Total Binding data Conservancy SequencePeptide Filed DR3 Motif 90.00 DHGAHLSLRIGLPVCA DR3 5.00 FHQAVLDPRVRGLYLDR3 45.00 PLGFFPDHQLDPAFG DR3 70.00 CLTRGRETVLEYLVS DR3 30.00RRSFGVEPSGSGHID DR3 55.00 GGVFLVDKNPHNTTE 35.0095 0.0790 DR3 55.00AFTFSPTYKAFLCKQ 35.0099 0.0035 DR3 65.00 AKLIGTDNSVVLSRK DR3 100.00NWSPWTHKVGNFTG DR3 65.00 AGPLEEELPRLADEG 35.0091 0.0022 DR3 100.00TKYLPLDKGIKPYYP 35.0094 −0.0017 DR3 55.00 LSWLSLDVSAAFYHI DR3 60.00VGPLTVNEKRRLKI 35.0093 * 2.2000 DR3 45.00 ESRLVDFSCFSAGN 35.0096 *2.6000 DR3 85.00 ASALYREALESPEHC DR3 45.00 LWGMDIDPYKEFGAS DR3 85.00NRRVAEDLNLGNLW 35.0092 0.1400 DR3 95.00 LCQVFADATPTGWGL 1280.14 0.0000DR3 35.00 LRPVAESRPPVSG 35.0101 −0.0017 DR3 90.00 FLLVLLDYCGMPVC 35.00900.0170 DR3 85.00 DNSVVLSRKYTSFPW DR3 90.0 AFSYMDDVVLGAKSV 1186.02 0.0130DR3 22 2

TABLE XX Population coverage with combined HLA Supertypes Phenotypicfrequency HLA-supertypes Caucasian N.A. Black Japanese Chinese HispanicAverage A2, A3 and B7 83.0 86.1 87.5 88.4 86.3 86.2 A2, A3, B7, A24, B4499.5 98.1 100.0 99.5 99.4 99.3 and A1 99.9 99.6 100.0 99.8 99.9 99.8 A2,A3, B7, A24 B44, A1, B27, B62, and B58

TABLE XXI HBV ANALOGS A2 A3 B7 1° Fixed A1 Super Super A24 Super AnchorAA Sequence Nomen. Motif Motif Motif Motif Motif Fixed Analog 10CILLLCLIFL N Y N N N No A 9 RMTGGVFLV VM2.V9 N Y N N N 1 A 9 LMPFVQWFVVM2.V9 N Y N N N 1 A 9 RLTGGVFLV VL2.V9 N Y N N N 1 A 9 GLCQVFADV L2.AV9N Y N N N 1 A 9 WLLRGTSFV IL2.V9 N Y N N N 1 A 9 NLGNLNVSV L2.IV9 N V NN N 1 A 9 YLPSALNPV VL2.AV9 N Y N N N 1 A 9 GLWIRTPPV VL2.AV9 N Y N N N1 A 9 RLSWPKFAV VL2.V9 N Y N N N 1 A 9 ILGLLGFAV VL2.AV9 N Y N N N 1 A 9RMLTIPQSV IM2.LV9 N Y N N N 1 A 9 SLDSWWTSV L2.LV9 N Y N N N 1 A 10FMLLLCLIFL IM2.L10 N Y N Y N 1 A 10 LMLQAGFFLV VM2.LV10 N Y N N N 1 A 10SMLSPFLPLV IM2.LV10 N Y N N N 1 A 10 LMLLDYQGMV VM2.LV10 N Y N N N 1 A10 FLGLSPTVWV VL2.LV10 N Y N N N 1 A 8 FPAAMPHL N N N N Y A 8 HPFAMPHL NN N N Y A 8 HPAAMPHI N N N N Y A 8 FMFSPTYK N N Y N N A 8 FVFSPTYK N N YN N A 9 FLLTRILTV L2.IV9 N Y N N N 1 A 9 ALMPLYACV L2.IV9 N Y N N N 1 A9 LLAQFTSAV L2.IV9 N Y N N N 1 A 9 LLPFVQWFV VL2.V9 N Y N N N 1 A 9FLLAQFTSV L2.AV9 N Y N N N 1 A 9 KLHLYSHPV L2.1V9 N Y N N N 1 A 9KLFLVSHPI N Y N N N No A 9 LLSSNLSWV L2.LV9 N Y N N N 1 A 9 FLLSLGIHVL2.LV9 N Y N N N 1 A 9 MMWYWGPSV M2.LV9 N Y N N N 1 A 9 VLQAGFFLV L2.LV9N Y N N N 1 A 9 PLLPIFFCV L2.LV9 N Y N N N 1 A 9 FLLPIFFCL N Y N N N NoA 9 VLLDYQGMV L2.LV9 N Y N N N 1 A 9 YMFDVVLGA N Y N N N No A 9GLLGWSPQV L2.AV9 N Y N N N 1 A 9 FPAAMPHLL N N N N Y A 9 HPFAMPHLL N N NN Y A 9 HPAAMPHLI N N N N Y A 9 FPVCAFSSA N N N N Y A 9 LPFCAFSSA N N NN Y A 9 LPVCAFSSI N N N N Y A 9 FPALMPLYA N N N N Y A 9 YPFLMPLYA N N NN Y A 9 YPALMPLYI N N N N Y A 9 FPSRGRLGL N N N N Y A 9 DPFRGRLGL N N NN Y A 9 DPSRGRLGI N N N N Y A 9 SMICSVVRR N N Y N N A 9 SVICSVVRR N N YN N A 9 KVGNFTGLK N N Y N N A 9 KVGNFTGRH N N Y N N A 9 WFFSQFSR N N Y NN A 9 SVNRPIDWK N N Y N N A 9 TLWKAGILK N N Y N N A 9 TLWKAGILR N N Y NN A 9 TMWKAGILY Y N Y N N A 9 TVWKAGILY N N Y N N A 9 RMYLHTLWK N N Y NN A 9 RVYLHTLWK N N Y N N A 9 AMTFSPTYK N N Y N N A 9 SVVRRAFPR N N Y NN A 9 SVVRRAFPK N N Y N N A 9 SAIXSVVRR N N Y N N A 9 LPVXAFSSA N N N NY A 10 FLLAQFTSAV L2.IV10 N Y N N N 1 A 10 YLFTLWKAGI N Y N N N No A 10YLLTLWKAGI N Y N N N No A 10 LLFYQGMLPV N Y N N N No A 10 LLLYQGMLPV N YN N N No A 10 LLVLQAGFFV L2.LV10 N Y N N N 1 A 10 ILLLCLIFLV L2.LV10 N YN N N 1 A 10 FPFCLAFSYM N N N N Y A 10 FPHCLAFSYI N N N N Y A 10FPARVTGGVF N N N N Y A 10 TPFRVTGGVF N N N N Y A 10 TPARVTGGVI N N N N YA 10 FPCALRFTSA N N N N Y A 10 GPFALRFTSA N N N N Y A 10 GPCALRFTSI N NN N Y A 10 FPAAMPHLLV N N N N Y A 10 HPFAMPHLLV N N N N Y A 10HPAAMPHLLI N N N N Y A 10 QMFTFSPTYK N N Y N N A 10 QVFTFSPTYK N N Y N NA 10 TMWKAGILYK N N Y N N A 10 TVWKAGILYK N N Y N N A 10 VMGGVFLVDK N NY N N A 10 VVGGVFLVDK N N Y N N A 10 SMLPETTVVR N N Y N N A 10SVLPETTVVR N N Y N N A 10 TMPETTVVRR N N Y N N A 10 TVPETTVVRR N N Y N NA 10 HTLWKAGILK N N Y N N A 10 HTLWKAGILR N N Y N N A 10 HMLWKAGILY Y NY N N A 10 HVLWKAGILY N N Y N N A 10 GMDNSVVLSR N N Y N N A 10GVDNSVVLSR N N Y N N A 10 GTFNSVVLSR N N Y N N A 10 YMFDVVLGAK N N Y N NA 10 MMWYWGPSLK N N Y N N A 10 MMWYWGPSLR N N Y N N A 9 ILLLXLIFL N Y NN N A 9 LLLXLIFLL N Y N N N A 9 LLXLIFLLV N Y N N N A 9 PLLPIFFXL N Y NN N A 9 ALMPLYAXI N Y N N N A 9 GLXQVFADA N Y N N N A 9 HISXLTFGR N N YN N A 9 FVLGGXRHK N N Y N N A 10 FILLLXLIFL N Y N N N A 10 ILLLXLIFLL NY N N N A 10 LLLXLIFLLV N Y N N N A 10 LLPIFFXLWV N Y N N N A 10QLLWFHISXL N Y N N N A 10 LLGXAANWIL N Y N N N A 10 TSAIXSVVRR N N Y N NA 10 GYRWMXLRRF N N N Y N A 10 GPXALRFTSA N N N N Y A 10 FPHXLAFSYM N NN N Y A 11 HMLWKAGILYK N N Y N N A 11 HVLWKAGILYK N N Y N N A 11SMLPETTVVRR N N Y N N A 11 SVLPETTVVRR N N Y N N A 11 GMDNSVVLSRK N N YN N A 11 GVDNSVVLSRK N N Y N N A 11 GTFNSVVLSRK N N Y N N A 8 MPLSYQHI NN N N Y A 8 LPIFFCLI N N N N Y A 8 SPFLLAQI N N N N Y A 8 YPALMPLI N N NN Y A 8 VPSALNPI N N N N Y A 9 LPIFFCLWI N N N N Y A 9 LPIHTAELI N N N NY A 10 VPFVQWFVGI N N N N Y A 11 NPLGFFPDHQI N N N N Y A 11 LPIHTAELLAIN N N N Y A 9 FLPSYFPSA L2.FY5.VA9 N Y N N N Rev3 A 10 VLHTLWKAGV-L2.IV10 N Y N N N 1 A 11 STLPETYVVRR N N Y N N A 9 YMDDVVLGV M2.AV9 N YN N N 1 A 9 FPIPSSWAF N N N N Y A 9 IPITSSWAF N N N N Y A 9 IPILSSWAF NN N N Y A 9 FPVCLAFSY N N N N Y A 9 FPHCLAFAY N N N N Y A 9 FPHCLAFSL NN N N Y A 9 IPIPMSWAF N N N N Y A 9 FPHCLAFAL N N N N Y A 10 FLPSZFFPSVN Y N N N No A 10 FLPSZFFPSV N Y N N N No A 9 IPFPSSWAF N N N N Y A 9IPIPSSWAI N N N N Y A 9 FPFCLAFSY N N N N Y A 9 FPHCLAFSI N N N N Y A 9FPHCLAFSA N N N N Y A 10 FQPSDYFPSV N Y N N N Rev A 9 VLLTRILTI N Y N NN A 9 FLYTRILTI N Y N N N A 9 FLLTYILTI N Y N N N A 9 FLLTRILYI N Y N NN A 11 FLPSDFFPSVR N N Y N N A 9 FLPSDFFPS N N N N N A 8 FLPSDFFP N N NN N A 10 FLPSDFFPSI L2.VI10 N Y N N N Rev A 10 FLPSDYFPSV N Y N N N No A12 VSFLPSDFFPSV N N N N A 10 YNMGLKFRQL N N N N N A 9 NMGLKYRQL N Y N YN No A 10 FLPS(X)YFPSV N N N N N A 10 FLPSD(X)FPSV N N N N N A 11FLPSDLLPSVR N N Y N N A 12 FLPSDFFPSVRD N N N N N A 12 LSFLPSDFFPSV N NN N N A 11 SFLPSDFFPSV N N N N N A 8 PSDFFPSV N N N N N A 9 FLMSYFPSV NY N N N No A 9 FLPSYFPSV L2.FY5.V9 N Y N N N 3 A 10 FLMSDYFPSV N Y N N NNo A 11 CILLLCLIFLL N Y N N N No A 10 FLPNDFFPSA L2.SN4.VA10 N Y N N NRev A 10 FLPDDFFPSA L2.SD4.VA10 N Y N N N Rev A 10 FLPNDFFPSV N Y N N NNo A 10 FLPSDFFPSA L2.VA10 N Y N N N Rev A 10 FLPDDFFPSV N Y N N N No A10 FLPADFFPSV N Y N N N No A 10 FLPVDFFPSV N Y N N N No A 10 FLPADFFPSIL2.SA4.VI10 N Y N N N Rev A 10 FLPVDFFPSI L2.SV4.VI10 N Y N N N Rev A 10FLPSDAFPSV N Y N N N No A 10 FLPSAFFPSV N Y N N N No A 10 FLPSDFAPSV N YN N N No A 10 FLPSDFFASV N Y N N N No A 10 FLPSDFFPAV N Y N N N No A 10FLASDFFPSV N Y N N N No A 10 FAPSDFFPSV LA2.V10 N Y N N N Rev A 10ALPSDFFPSV N Y N N N No A 10 YLPSDFFPSV N Y N N N No A 10 FMPSDFFPSVLM2.V10 N Y N N N 1 A 10 FLKSDFFPSV N Y N N N No A 10 FLPSEFFPSV N Y N NN No A 10 FLPSDFYPSV N Y N N N No A 10 FLPSDFFKSV N Y N N N No A 10FLPSDFFPKV N Y N N N No A 10 FLPSDFFPSV(CONH2) N N N N N Amidated 10VLEYLVSFGV(NH2) N N N N N Amidaled 17 ATVELLSFLPSDFFPSV-NH2 N N N N NAmidaled 16 TVELLSFLPSDFFPSV-NH2 N N N N N Amidated 15VELLSFLPSDFFPSV-NH2 N N N N N Amidated 14 ELLSFLPSDFFPSV-NH2 N N N N NAmidated 13 LLSFLPSDFFPSV-NH2 N N N N N Amidated 12 LSFLPSDFFPSV-NH2 N NN N N Amidated 11 SFLPSDFFPSV-NH2 N N N N N Amidated 10 FLPSDFFPSV-NH2 NN N N N Amidated 9 LPSDFFPSV-NH2 N N N N N Amidated 8 PSDFFPSV-NH2 N N NN N Amidated 9 FLPSDFFPS-NH2 N N N N N Amidated 8 FLPSDFFP-NH2 N N N N NAmidated 7 FLPSDFF-NH2 N N N N N Amidated 10 ALPSDFFPSV-NH2 N N N N NAmidated 10 SLNFLGGTTV(NH2) N N N N N Amidated 11 FLPSDFFPSVR-NH2 N N NN N Amidated 9 ALFKDWEEL N Y N N N A 9 VLGGSRHKL N Y N N N A 9 KIKESFRKLN Y N N N A 9 ALMPLYASI N Y N N N A 9 FLSKQYLNL N Y N N N A 9 LLGSAANWIN Y N N N A 9 NLNNLNVSI N Y N N N A 9 IIKKSEQFV N Y N N N A 9 ALSLIVNLLN Y N N N A 9 RIPRTPRSV N Y N N N A 237

TABLE XXII Discreet substitutions improve the B7 supertype bindingcapacity and degeneracy of peptide ligands. Binding (IC₅₀ nM) Source 1 23 4 5 6 7 8 9 B*0701 B*3501 B*5101 B*5301 B*5401 x-rxn HBV ENV I P I P SS W A F 42 2.6 2.3 12 2970 4 313 F P I P S S W A F 24 1.2 305 1.7 105 5I P I P S S W A I 31 54 15 24 7.7 5 HBV POL 541 F P H C L A F S Y — 1483 17 503 3 F P H C L A F A L 25 2.7 28 5.0 24 5 F P H C L A F S L 742.4 4.5 15 7.7 5 F P F C L A F S Y — 6.5 27 4.8 5.1 4 F P H C L A F S I675 29 6.3 3.8 1.0 4 F P H C L A F S A 3667 6.5 250 137 0.6 4 HCV Core168 L P G C S F S I F 28 90 100 114 6897 4 F P G C S F S I F 19 1.6 1323.2 67 5 MAGE2 170 V P I S H L Y I L 22 171 96 238 3175 4 F P I S H L YI L 16 7.3 6.4 7.0 28 5 MAGE3 196 M P K A G L L I I 940 5039 393 90 2483 F P K A G L L I I 162 1303 5.8 60 150 4 M P F A G L L I I 229 1.0 0.92.3 0.27 5

TABLE XXIII Sets of preferred epitopes restricted by class I and classII molecules can be selected for inclusion in an HBV-specific vaccine.Table XXIII lists as a matter of example one such set of epitopes.Peptide Sequence Protein restriction A) Class I restricted epitopes924.07 FLPSDFFPSV core 18 A2 777.03 FLLTRILTI env 183 A2 927.15ALMPLYACI pol 642 A2 1013.01 WLSLLVPFV env 335 A2 1090.14 YMDDVVLGA pol538 A2/A1 1168.02 GLSRYVARL pol 455 A2 927.11 FLLSLGIHL pol 562 A21069.10 LLPIFFCLWV env 378 A2 1069.06 LLVPFVQWFV env 338 A2 1147.16HTLWKAGILYK pol 149 A3/A1 1083.01 STLPETTVVRR core 141 A3 1069.16NVSIPWTHK pol 47 A3 1069.20 LVVDFSQFSR pol 388 A3 1090.10 QAFTFSPTYK pol665 A3 1090.11 SAICSVVRR pol 531 A3 1142.05 KVGNFTGLY pol 629 A3/A11147.05 FPHCLAFSYM pol 530 B7 988.05 LPSDFFPSV core 19 B7 1145.04IPIPSSWAF env 313 B7 1147.02 HPAAMPHLL pol 429 B7 26.0570 YPALMPLYACIpol 640 B7 1147.04 TPARVTGGVF pol 354 B7 1.0519 DLLDTASALY core 419 A12.0239 LSLDVSAAFY pol 1000 A1 1039.06 WMMWYWGPSLY env 359 A1 20.0269RWMCLRRFII env 236 A24 20.0136 SWLSLLVPF env 334 A24 20.0137 SWWTSLNFLenv 197 A24 13.0129 EYLVSFGVWI core 117 A24 1090.02 AYRPPNAPI core 131A24 13.0073 WFHISCLTF core 102 A24 20.0271 SWPKFAVPNL pol 392 A241069.23 KYTSFPWLL pol 745 A24 2.0181 LYSHPIILGF pol 492 A24 B) Glass IIrestricted epitopes F107.03 LQSLTNLLSSNLSWL pol 412 DR supermotif1298.06 KQAFTFSPTYKAFLC pol 664 1280.06 AGFFLLTRILTIPQS env 180 1280.09GTSFVYVPSALNPAD pol 774 CF-08 VSFGVWIRTPPAYRPPNAPI core 120 27.0281RHYLHTLWKAGILYK pol 145 1186.15 LVPFVQWFVGLSPTV env 339 1280.15LHLYSHPIILGFRKI pol 501 F107.04 PFLLAQFTSAICSVV pol 523 1298.04KQCFRKLPVNRPIDW pol 618 1298.07 AANWILRGTSFVYVP pol 767 857.02PHHTALRQAILCWGELMTLA core 50 1280.14 LCQVFADATPTGWGL pol 694 DR3 motif35.0096 ESRLVVDFSQFSRGN pol 385 35.0093 VGPLTVNEKRRLKLI pol 96 1186.27SSNLSWLSLDVSAAF pol 420 1186.18 NLSWLSLDVSAAFYH pol 442

TABLE 1 POSITION POSITION POSITION 3 (Primary C Terminus 2 (PrimaryAnchor) Anchor) (Primary Anchor) SUPERMOTIF A1 TI LVMS FWY A2 LIVM ATQIV MATL A3 VSMA TLI RK A24 YF WIVLMT FI YWLM B7 P VILF MWYA B27 RHK FYLWMI B44 E D FWYLIMVA B58 ATS FWY LIV B62 QL IVMP FWY MIV MOTIFS A1 TSM YA1 DE AS Y A3 LMVISATF CGD KYR HFA A11 VTMLISAGN CDF K RYH A24 YFWM FLIWA2.1 LM VQIAT V LIMAT A*3101 MVT ALIS R K A*3301 MVALF IST RK A*6801 AVTMSLI RK B*0702 P LMF WYAIV B*3501 P LMFWY IVA B51 P LIVF WYAM B*5301 PIMFWY ALV B*5401 P ATIV LMFWYBolded residues are preferred, italicized residues are less preferred: Apeptide is considered motif-bearing if it has primary anchors at eachprimary anchor position for a motif or supermotif as specified in theabove table.

1. A peptide composition of less than 100 amino acid residues comprisinga peptide epitope useful for inducing an immune response againsthepatitis B virus (HBV) said epitope (a) having an amino acid sequenceof about 8 to about 13 amino acid residues that have at least 65%identity with a native amino acid sequence for HBV, and, (b) binding toat least one MHC class I HLA allele with a dissociation constant of lessthan about 500 nM.
 2. The composition of claim 1, further wherein saidpeptide has at least 77% identity with a native HBV amino acid sequence.3. The composition of claim 1, further wherein said peptide has 100%identity with a native HBV amino acid sequence.
 4. The composition ofclaim 1, further wherein said peptide is one of those peptides describedin Tables VI-XVII or XXI.
 5. The composition of claim 4, further whereinsaid peptide is one of the peptides designated as being from theenvelope region of HBV.
 6. The composition of claim 4, further whereinsaid peptide is one of the peptides designated as being from thepolymerase region of HBV.
 7. The composition of claim 4, further whereinsaid peptide is one of the peptides designated as being from the proteinX region of HBV.
 8. The composition of claim 4, further wherein saidpeptide is one of the peptides designated as being from the nucleocapsidcore region of HBV.
 9. A composition of less than 100 amino acidresidues comprising a peptide epitope useful for inducing an immuneresponse against hepatitis B virus (HBV) said peptide (a) having anamino acid sequence of about 8 to about 13 amino acid residues and (b)bearing one of the HLA motifs set out in Tables I and II.
 10. Thecomposition of claim 9, further wherein said peptide is one of thosepeptides described in Table VI or Table XXI bearing an HLA A1supermotif.
 11. The composition of claim 9, further wherein said peptideis one of those peptides described in Table VII or Table XXI bearing anHLA A2 supermotif.
 12. The composition of claim 9, further wherein saidpeptide is one of those peptides described in Table VIII or Table XXIbearing an HLA A3 supermotif.
 13. The composition of claim 9, furtherwherein said peptide is one of those peptides described in Table IX orTable XXI bearing an HLA A24 supermotif.
 14. The composition of claim 9,further wherein said peptide is one of those peptides described in TableX or Table XXI bearing an HLA B7 supermotif.
 15. The composition ofclaim 9, further wherein said peptide is one of those peptides describedin Table XI bearing an HLA B27 supermotif.
 16. The composition of claim9, further wherein said peptide is one of those peptides described inTable XII bearing an HLA B44 supermotif.
 17. The composition of claim 9,further wherein said peptide is one of those peptides described in TableXIII bearing an HLA B58 supermotif.
 18. The composition of claim 9,further wherein said peptide is one of those peptides described in TableXIV bearing an HLA B62 supermotif.
 19. The composition of claim 9,further wherein said peptide is one of those peptides described in TableXV bearing an HLA A1 motif.
 20. The composition of claim 9, furtherwherein said peptide is one of those peptides described in Table XVIbearing an HLA A3 motif.
 21. The composition of claim 9, further whereinsaid peptide is one of those peptides described in Table XVI bearing anHLA A11 motif.
 22. The composition of claim 9, further wherein saidpeptide is one of those peptides described in Table XVII bearing an HLAA24 motif.
 23. The composition of claim 9, further wherein said peptideis one of those peptides described in Table VII bearing an HLA A2.1motif wherein the epitope is numbered from an amino terminal to carboxylterminal orientation relative to the peptide, with the proviso that thepeptide does not bear L or M at position 2 and V at C-terminal position9 of a 9 amino acid peptide.
 24. An analog of an HBV peptide of lessthan 100 amino acid residues in length that bears an HLA binding motif,the analog bearing the same HLA binding motif as the peptide butcomprising at least one anchor residue that is different from that ofthe peptide.
 25. The composition of claim 24, further wherein saidpeptide is an analog of a peptide described in Table VI bearing an HLAA1 supermotif.
 26. The composition of claim 24, further wherein saidpeptide is an analog of a peptide described in Table VII bearing an HLAA2 supermotif.
 27. The composition of claim 24, further wherein saidpeptide is an analog of a peptide described in Table VIII bearing an HLAA3 supermotif.
 28. The composition of claim 24, further wherein saidpeptide is an analog of a peptide described in Table IX bearing an HLAA24 supermotif.
 29. The composition of claim 24, further wherein saidpeptide is an analog of a peptide described in Table X bearing an HLA B7supermotif.
 30. The composition of claim 24, further wherein saidpeptide is an analog of a peptide described in Table XI bearing an HLAB27 supermotif.
 31. The composition of claim 24, further wherein saidpeptide is an analog of a peptide described in Table XII bearing an HLAB44 supermotif.
 32. The composition of claim 24, further wherein saidpeptide is an analog of a peptide described in Table XIII bearing an HLAB58 supermotif.
 33. The composition of claim 24, further wherein saidpeptide is an analog of a peptide described in Table XIV bearing an HLAB62 supermotif.
 34. The composition of claim 24, further wherein saidpeptide is an analog of a peptide described in Table XV bearing an HLAA1 motif.
 35. The composition of claim 24, further wherein said peptideis an analog of a peptide described in Table XVI bearing an HLA A3motif.
 36. The composition of claim 24, further wherein said peptide isan analog of a peptide described in Table XVI bearing an HLA A11 motif.37. The composition of claim 24, further wherein said peptide is ananalog of a peptide described in Table XVII bearing an HLA A24 motif.38. The composition of claim 24, further wherein said peptide is ananalog of a peptide described in Table VII bearing an HLA A2.1 motif.39. The composition of claim 24, wherein said peptide is an analog of apeptide described in Table XVIII or Table XIX comprising at HLA class IImotif.
 40. A composition of less than 100 amino acid residues comprisinga peptide epitope useful for inducing an immune response againsthepatitis B virus (HBV) said peptide (a) having an amino acid sequenceof about 9 to about 25 amino acid residues that have at least 65%identity with a native amino acid sequence for HBV and (b) binding to atleast one MHC class II HLA allele with a dissociation constant of lessthan about 1000 nM.
 41. The composition of claim 40, further whereinsaid peptide has at least 77% identity with a native HBV amino acidsequence.
 42. The composition of claim 40, further wherein said peptidehas 100% identity with a native HBV amino acid sequence.
 43. Thecomposition of claim 40, further wherein said peptide is one of thosepeptides described in Table XVIII or Table XIX.
 44. A peptidecomposition of less than 100 amino acid residues, said compositioncomprising an epitope useful for inducing an immune response againsthepatitis B virus (HBV) said epitope (a) having an amino acid sequenceof about 9 to about 25 amino acid residues and (b) bearing one of theclass II HLA motifs set out in Table III.
 45. The composition of claim44, further wherein said peptide is one of those peptides described inTable XVIII or XIX.
 46. A composition that comprises an isolated nucleicacid sequence that encodes one of the peptides set out in Tables VI-XIXor XX or XXIII.
 47. A composition that comprises at least two peptidesat least one of said at least two peptides selected from Tables VI-XIXor XXI or XXIII.
 48. A composition of claim 47 wherein two or more ofthe at least two peptides are depicted in Tables VI-XIX or XXI or XXIII.49. A composition that comprises at least one nucleic acid sequence,that encodes the peptides of claim
 47. 50. The composition of claim 47wherein each of said at least two peptides are encoded by a nucleic acidsequence, wherein each of the nucleic acid sequences are located on asingle vector.
 51. A peptide composition of less than 100 amino acidresidues, said composition comprising an epitope useful for inducing animmune response against hepatitis B virus (HBV) said epitope having atleast one of the amino acid sequences set out in Table XXIII.
 52. Amethod for inducing a cytotoxic T cell response to HBV in a mammalcomprising administering to said mammal at least one peptide from TablesVI to XIX or Table XXI.
 53. A vaccine for treating HBV infection thatinduces a protective immune response, wherein said vaccine comprises atleast one peptide selected from Tables VI to Table XIX or Table XXI in apharmaceutically acceptable carrier.
 54. A vaccine for preventing HBVinfection that induces a protective immune response, wherein saidvaccine comprises at least one peptide selected from Tables VI to XIX orTable XX in a pharmaceutically acceptable carrier.
 55. A method forinducing a cytotoxic T cell response to HBV in a mammal, comprisingadministering to said mammal a nucleic acid sequence encoding a peptideselected from Tables VI to XIX or Table XXI.
 56. A kit for a vaccine fortreating or preventing HBV infection, wherein the vaccine induces aprotective immune response, said vaccine comprising at least one peptideselected from Tables VI to XIX or Table XXI in a pharmaceuticallyacceptable carrier and instructions for administration to a patient. 57.A method for monitoring an immune response to HBV or an epitope thereofin a patient having a known HLA-type, the method comprising incubating aT lymphocyte sample from the patient with a peptide selected from TablesVI to XIX or Table XXI, which peptide binds a motif corresponding to atleast one HLA allele present in said patient, and detecting the presenceof a T lymphocyte that recognizes the peptide.
 58. The method of claim57, wherein the peptide comprises a tetrameric complex.